U.S. patent application number 12/228710 was filed with the patent office on 2010-01-21 for attenuation of encephalitogenic alphavirus and uses thereof.
Invention is credited to Ilya V. Frolov, Elena Frolova, Scott C. Weaver.
Application Number | 20100015179 12/228710 |
Document ID | / |
Family ID | 41530482 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100015179 |
Kind Code |
A1 |
Frolov; Ilya V. ; et
al. |
January 21, 2010 |
Attenuation of encephalitogenic alphavirus and uses thereof
Abstract
The present invention is drawn to generating attenuated and less
cytopathic forms of New World alphaviruses that can be used in
immunogenic compositions as vaccines against both Old and New World
alphaviruses. In this regard, the present invention discloses that
the N-terminal, .about.35-aa-long peptide of VEEV, EEEV and, most
likely, of WEEV capsid proteins plays the most critical role in the
downregulation of cellular transcription and development of
cytopathic effect. The identified, VEEV-specific peptide,
C.sub.VEE30-68, includes two domains with distinguished functions.
The integrity of both domains determines not only the intracellular
distribution of C.sub.VEE, but is also essential for direct capsid
function in the inhibition of transcription. The replacement of the
N-terminal fragment of C.sub.VEE by its SINV-specific counterpart
in VEEV TC-83 genome does not affect virus replication in vitro,
but makes it less cytopathic and more attenuated in vivo.
Inventors: |
Frolov; Ilya V.; (Galveston,
TX) ; Frolova; Elena; (Galveston, TX) ;
Weaver; Scott C.; (Galveston, TX) |
Correspondence
Address: |
Benjamin Aaron Adler;ADLER & ASSOCIATES
8011 Candle Lane
Houston
TX
77071
US
|
Family ID: |
41530482 |
Appl. No.: |
12/228710 |
Filed: |
August 15, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60964969 |
Aug 16, 2007 |
|
|
|
Current U.S.
Class: |
424/205.1 ;
435/236 |
Current CPC
Class: |
C12N 2770/36161
20130101; A61K 39/12 20130101; C12N 7/00 20130101; A61P 31/12
20180101; A61K 2039/5254 20130101; C12N 2770/36122 20130101; C07K
14/005 20130101; A61P 31/14 20180101; C12N 2770/36134 20130101 |
Class at
Publication: |
424/205.1 ;
435/236 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C12N 7/04 20060101 C12N007/04; A61P 31/12 20060101
A61P031/12 |
Goverment Interests
FEDERAL FUNDING LEGEND
[0002] This invention was produced using funds obtained through
National Institutes of Health grant AI057156 and Public Health
Service grant AI050537. Consequently, the Federal government has
certain rights in this invention.
Claims
1. A method of attenuating New World encephalitogenic alphavirus
comprising: mutating one or more than one amino acids in the amino
terminal region of the capsid protein of the alphavirus; or
replacing the entire capsid protein or amino terminal region of the
capsid protein of the alphavirus by capsid protein or amino
terminal region of a less pathogenic Old World alphavirus.
2. The method of claim 1, wherein said mutation comprises point
mutations in the amino terminal of the capsid protein.
3. The method of claim 1, wherein the amino acid(s) mutated or
replaced comprises amino acids 33-68 of Venezuelan Equine
Encephalitis virus capsid protein, amino acids 36-72 of Eastern
Equine Encephalitis virus capsid protein or amino acids 36-72 of
Western Equine Encephalitis virus capsid protein.
4. The method of claim 1, wherein the Old World alphavirus is
Sindbis, Semliki Forest, Ross River, Aura and other antigenically
related viruses.
5. The method of claim 1, wherein the attenuated New World,
encephalitogenic alphavirus is capable of replicating in vitro but
cannot cause disease in animals and in immunized individuals.
6. An immunogenic composition, comprising: the attenuated New World
encephalitogenic alphavirus of claim 1.
7. The immunogenic composition of claim 6, wherein the New World
encephalitogenic alphavirus comprises mutations of one or more than
one amino acids in the amino terminal of the capsid protein of the
alphavirus, deletion of entire capsid protein or deletion of amino
terminal of the capsid protein.
8. The immunogenic composition of claim 7, wherein the deleted
capsid protein or amino terminal of the capsid protein is replaced
by capsid protein or amino terminal of the capsid protein of less a
pathogenic Old World alphavirus.
9. The immunogenic composition of claim 7, wherein the amino
acid(s) mutated or replaced comprises amino acids 33-68 of
Venezuelan Equine Encephalitis virus capsid protein, amino acids
36-72 of Eastern Equine Encephalitis virus capsid protein or amino
acids 36-72 of Western Equine Encephalitis virus capsid
protein.
10. The immunogenic composition of claim 7, wherein the Old World
alphavirus is Sindbis, Semliki Forest, Ross River, Aura and other
antigenically related viruses.
11. A method of preventing an infection caused by Old and New World
encephalitogenic alphavirus in a subject, comprising: administering
an immunologically effective amount of the immunogenic composition
of claim 7 to said subject.
12. The method of claim 11, wherein the New World encephalitogenic
alphavirus is Venezuelan Equine Encephalitis virus, Western Equine
Encephalitis virus or Western Equine Encephalitis virus.
13. The method of claim 11, wherein the Old World alphavirus is
Sindbis virus, Semliki Forest virus, Ross River virus or Aura
virus.
14. The method of claim 11, wherein the immunogenic composition is
administered subcutaneously or intramuscularly.
15. The method of claim 11, wherein the subject is a human or an
animal, wherein said subject is a healthy subject or a subject who
is likely to be exposed to the alphavirus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This nonprovisional application claims benefit of priority
of U.S. Ser. No. 60/964,969, filed Aug. 16, 2007, now abandoned,
and is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the field of
virology and vaccine development. More specifically, the present
invention provides a method to attenuate encephalitogenic
alphaviruses including but not limited to VEEV, EEEV and WEEV. The
attenuated phenotype is irreversible and thus, can be effective as
human and/or veterinary vaccines in immunogenic composition(s).
[0005] 2. Description of the Related Art
[0006] The Alphavirus genus in the Togaviridae family includes a
number of important human and animal pathogens (15). Alphaviruses
are currently classified into 6 antigenic complexes and are widely
distributed both in the New and the Old World. They are efficiently
transmitted by mosquitoes, in which they cause a persistent,
lifelong infection that does not noticeably affect biological
functions of the vectors. In vertebrates, the infection is acute
and characterized by high-titer viremia, rash and fever and
encephalitis until the death of the infected host or clearance of
the virus by the immune system. The encephalitogenic alphaviruses,
including Venezuelan (VEEV), eastern (EEEV) and western equine
encephalitis (WEEV) viruses, represent a continuous public health
threat in the U.S. (40, 47-49). They circulate in the Central,
South and North Americas and have an ability to cause fatal disease
in humans and horses. During VEEV epizootics, equine mortality can
reach 83%, and, in humans, this virus produces a severe temporary
immunodeficiency and a greatly debilitating and sometimes fatal
disease (41). The overall mortality rate is below 1%, but the
neurological disease, including disorientation, ataxia, mental
depression, and convulsions, can be detected in up to 14% of all
infected individuals, especially children (23). Sequelae of
VEEV-related clinical encephalitis in humans are also described
(10, 27).
[0007] The VEEV genome is represented by a single-stranded RNA
molecule of positive polarity of almost 12-kb. It mimics the
structure of cellular mRNA, in which it contains a Cap at the 5'
terminus and a poly(A) tail at the 3' end of the RNA. VEEV genome
has been cloned in a cDNA form (24) that allows a wide variety of
genetic manipulation to be undertaken.
[0008] The current experimental vaccine against VEEV infection was
developed four decades ago by serial passaging of the virulent,
subtype IAB Trinidad Donkey (TRD) VEEV strain in guinea pig heart
cell cultures (3). Presently, TC-83 is still the only available
vaccine for laboratory workers and military personnel. Over 8,000
humans have been vaccinated during the past 4 decades (2, 6, 36),
and the cumulative data unambiguously demonstrated that nearly 40%
of vaccinated people develop a disease with some symptoms typical
of natural VEEV infection, including a febrile, systemic illness
and other adverse effects (2, 3, 21). No effective antivirals have
been developed against this virus as well.
[0009] In spite of the continuous threat of VEEV epidemics, the
biology of this virus has been studied less intensively than that
of other, less pathogenic alphaviruses, such as Sindbis (SINV) and
Semliki Forest (SFV) viruses. This situation can be partially
explained by the fact that for a long time, it was believed that
the latter viruses represent excellent models for studying the
mechanism of alphavirus replication, virus-host interactions and
encephalitis development (14). However, very strong differences in
pathogenesis and the severity of the caused diseases suggest that
this may not exactly be the case. Moreover, the results from recent
comparative studies with the Old World alphaviruses (SINV and SFV)
and the New World alphaviruses (VEEV and EEEV) (1, 9, 11-13, 35,
45) demonstrated that both of these groups have developed the
ability to interfere with cellular transcription and use it as a
means of downregulating cellular antiviral reactions. However, the
mechanism of transcription inhibition appears to be fundamentally
different, and while the Old World alphaviruses use nsP2 to inhibit
cellular transcription (11), the more encephalitogenic VEEV and
EEEV use their capsid protein for the same function (1, 12).
Expression of the latter protein by different vectors is sufficient
for induction of cell death and cytopathic effect (CPE) in tissue
culture, and this effect strongly correlates with the inhibition of
transcription of cellular messenger and ribosomal RNAs. Moreover,
the replacement of structural genes in VEEV by those derived from
SINV made the chimeric virus strongly less cytopathic and incapable
of interfering with the development of an antiviral reaction
developing in the cells having no defect in IFN-a/b induction and
signaling (12).
[0010] Despite this, prior art is deficient in an immunogenic
composition(s) that will prevent and treat infection caused by
encephalitogenic alphavirus. The current invention fulfils this
long standing need in the art.
SUMMARY OF THE INVENTION
[0011] In one embodiment of the present invention, there is
provided a method of attenuating a New World encephalitogenic
alphavirus comprising: mutating one or more than one amino acids in
the amino terminus of the capsid protein of the alphavirus or
replacing the entire capsid protein or amino terminus of the capsid
protein of the alphavirus by capsid protein or amino terminus of
less pathogenic Old World alphavirus.
[0012] In a related embodiment of the present invention, there is
provided an immunogenic composition, comprising the attenuated New
World encephalitogenic alphavirus generated by the method described
supra.
[0013] In another related embodiment of the present invention,
there is provided a method of preventing an infection caused by Old
and New World encephalitogenic alphavirus in a subject, comprising:
administering an immunologically effective amount of the
immunogenic composition described supra to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The appended drawings have been included herein so that the
above-recited features, advantages and objects of the invention
will become clear and can be understood in detail. These drawings
form a part of the specification. It is to be noted, however, that
the appended drawings illustrate preferred embodiments of the
invention and should not be considered to limit the scope of the
invention.
[0015] FIGS. 1A-1F show the effect of expression of capsid mutants
on cellular transcription and cell growth and the ability of cells
to form PurR foci. FIGS. 1A and 1B are schematic representations of
VEEV replicons expressing GFP or mutated capsids. Arrows indicate
positions of the subgenomic promoters. Replicons expressed either
CVEE with mutated protease (mutCVEE) (FIG. 1A) or CVEE having no
mutations in protease domain (FIG. 1B). VEErepL/CVEEfrsh/Pac was
included in FIGS. 1B, 1D and 1F as an additional control. Different
dilutions of the electroporated cells were seeded into 100-mm
tissue culture dishes and Puromycin selection performed. PurR cell
colonies were stained with crystal violet at 4-9 days
post-transfection, depending on their growth rates. The results are
presented in colony-forming units (CFU) per mg of RNA used for
transfection. The ranges indicate variations between the
experiments. FIGS. 1C and 1D compare growth of the cells
transfected with VEEV replicons expressing GFP, and different
capsids. Equal numbers of cells were seeded into 6-well Costar
plates. Puromycin selection (10 mg/ml) was performed between 6 and
48 h post transfection. Then cells were incubated in puromycin-free
media, and viable cells were counted at the indicated times. The
data were normalized on the number of viable adherent cells
determined at 6 h post transfection. FIGS. 1E and 1F show analysis
of cellular transcription. RNA labeling was performed with
[.sup.3H]uridine at 24 post transfection for 2 h. RNA samples were
analyzed by gel electrophoresis. For quantitative analysis, the
aliquots of the RNA samples used for the gel were washed on the
Whatman 3 MM filters with TCA and the radioactivity was measured by
liquid scintillation counting. Error bars indicate variations
between parallel samples.
[0016] FIGS. 2A-2C show effect of the C-terminal deletions in
capsid on cellular transcription and cell viability. FIG. 2A is a
schematic representation of VEEV genome-based replicons expressing
the amino terminal fragments of C.sub.VEE, fused with GFP, and
analysis of their ability to establish persistent replication and
develop Pur.sup.R foci. Arrows indicate the positions of the
subgenomic promoters. Numbers indicate the last capsid-specific
amino acid. FIG. 2B compares growth of cells carrying VEEV
replicons expressing GFP or indicated fusions. FIG. 2C shows
inhibition of transcription in the BHK-21 cells transfected with
VEEV replicons expressing indicated proteins. Cells were
electroporated by 5 mg of the in vitro-synthesized RNAs. At 24 h
post-transfection, cellular RNAs were labeled with [.sup.3H]uridine
in the absence of ActD for 2 h and analyzed by RNA gel
electrophoresis. Quantitative analysis of residual cellular
transcription was performed as described in FIG. 1. Error bars
indicate variations between parallel samples.
[0017] FIGS. 3A-3D show effect of the N-terminal VEEV capsid
fragment on cellular transcription and cell viability. FIG. 3A
shows sequence alignment of C.sub.VEE30-68 peptide (SEQ ID NO: 1)
with the corresponding capsid fragment of other alphaviruses. VEEV,
Venezuelan equine encephalitis virus; EEEV, eastern equine
encephalitis virus (SEQ ID NO: 2); SINV, Sindbis virus (SEQ ID NO:
3); SFV, Semliki Forest virus (SEQ ID NO: 4). Helix I sequences are
indicated in red. Residues identical to those in the VEEV sequence
are indicated by dashes. Stars indicate positions of the deletions
introduced for better alignment of the sequences. FIG. 3B is a
schematic representation of VEEV genome-based replicons expressing
the amino terminal fragments of C.sub.VEE, fused with GFP, and
analysis of their ability to establish persistent replication and
develop PurR foci. Arrows indicate the positions of the subgenomic
promoters. Numbers indicate the last capsid-specific amino acid.
The results are presented in colony-forming units (CFU) per mg of
RNA used for transfection. The ranges indicate variations between
the experiments. FIG. 3C compares growth of the cells transfected
with VEE replicons expressing GFP, and different capsids. The data
were normalized on the number of viable adherent cells determined
at 6 h post transfection. FIG. 3D shows analysis of cellular
transcription. RNA labeling was performed with [3H]uridine at 24 h
post transfection for 2 h and RNA samples were analyzed by gel
electrophoresis. Quantitative analysis of residual cellular
transcription was performed as described in FIG. 1. Error bars
indicate variations between parallel samples.
[0018] FIGS. 4A-4D show comparison of the effects of C.sub.VEE and
C.sub.VEEGFP fusions expression on cellular transcription and cell
viability. FIG. 4A is a schematic representation of VEEV
genome-based replicons expressing C.sub.VEE and C.sub.VEEGFP
fusion. Analysis of the replicons' ability to establish persistent
replication and develop Pur.sup.R foci. Arrows indicate the
positions of the subgenomic promoters. FIG. 4B shows growth of the
cells transfected with VEE replicons expressing either GFP or
C.sub.VEE. The data were normalized on the number of viable
adherent cells determined at 6 h post transfection. FIG. 4C shows
analysis of cellular transcription. RNA labeling was performed with
[.sup.3H]uridine at 24 h post transfection for 2 h. RNA samples
were analyzed by gel electrophoresis. Quantitative analysis of
residual cellular transcription was performed as described in FIG.
1. Error bars indicate variations between parallel samples. FIG. 4D
shows analysis of the GFP-containing proteins expressed by
indicated replicons. Cell lysates were prepared at 20 h post
transfection and analyzed by western blotting using GFP-specific
antibody.
[0019] FIGS. 5A-5B show effect of the N-terminal deletions in
capsid on cellular transcription and cell viability. FIG. 5A is a
schematic representation of VEEV genome-based replicons expressing
the deleted forms of capsid fused with GFP, and analysis of their
ability to establish persistent replication and develop PurR foci.
Arrows indicate the positions of the subgenomic promoters. Numbers
indicate the first amino acid of CVEE after deletion. In all of the
constructs, the initiating capsid AUG was present in its natural
position. FIG. 5B compares growth of the cells carrying VEEV
replicons expressing GFP or indicated fusions.
[0020] FIGS. 6A-6C compare effect of the expression of C.sub.VEE
peptides fused with GFP or GFP.sub.NLS on cell viability and
cellular transcription. FIG. 6A is a schematic representation of
VEEV genome-based replicons expressing the deleted forms of capsid
fused with GFP, and analysis of their ability to establish
persistent replication and develop Pur.sup.R foci. Arrows indicate
the positions of the subgenomic promoters. In all of the
constructs, the initiating AUG was created upstream of the studied
peptide. FIG. 6B shows growth of the cells carrying VEEV replicons
expressing indicated fusions. FIG. 6C shows analysis of cellular
transcription. RNA labeling was performed with [.sup.3H]uridine at
24 h post transfection for 2 h. RNA samples were analyzed by gel
electrophoresis. Quantitative analysis of residual cellular
transcription was performed as described in FIG. 1. Error bars
indicate variations between parallel samples.
[0021] FIGS. 7A-7C compares effects of C.sub.VEE30-68 and
C.sub.EEE33-71 peptides, fused with GFP.sub.NLS, on cellular
transcription and cell viability. FIG. 7A is a schematic
representation of VEEV genome-based replicons expressing GFP or
fusion proteins. The initiating AUG was created upstream of the
studied peptides. Analysis of the replicons' ability to establish
persistent replication and develop Pur.sup.R foci. FIG. 7B shows
growth of the cells transfected with VEE replicons expressing
indicated proteins. The data were normalized on the number of
viable adherent cells determined at 6 h post transfection. FIG. 7C
shows analysis of cellular transcription. RNA labeling was
performed with [.sup.3H]uridine at 24 h post transfection for 2 h.
RNA samples were analyzed by gel electrophoresis. Quantitative
analysis of residual cellular transcription was performed as
described in FIG. 1. Error bars indicate variations between
parallel samples.
[0022] FIGS. 8A-8B show intracellular distribution of different
C.sub.VEEGFP fusions. In FIG. 8A, BHK-21 cells were transfected
with the replicons expressing C.sub.VEEGFP (a), C.sub.VEED35-47GFP
(b) and C.sub.VEEfrshGFP (c) proteins, and the intracellular
distribution of the fusions was analyzed at 12 h post transfection.
The high magnification images of the nuclei-containing cell
fragments were acquired on the confocal microscope. In FIG. 8B,
BHK-21 cells were transfected with VEErepL/C.sub.VEEGFP/Pac, and,
at 12 h post transfection, cells were permeabalized with 0.5%
Triton X-100, stained with MAb414 antibodies and AlexaFluor
546-labeled secondary antibodies and analyzed on a confocal
microscope. FIG. 8B: (a) Distribution of C.sub.VEE/GFP on the
nuclear membrane, (b) MAb414 staining of the same cell and (c)
overlay of the images (fragment of the image indicated on panels a
and b is shown).
[0023] FIGS. 9A-9C show replication of the VEEV TC-83 having
mutated capsid protein. FIG. 9A is a schematic representation of
the viral genomes. In VEEV/CSIN1, the natural N-terminal fragment,
located upstream of the protease domain (aa 1-110), was replaced by
its SINV-specific counterpart (aa 1-98), indicated by black box. In
VEEV/Cfrsh, the capsid contained a frame-shift mutations that
changed the peptide between aa 57 and 86. In FIG. 9B, BHK-21 cells
were electroporated with 5 mg of in vitro-synthesized RNAs.
One-fifth of the samples were seeded into 35-mm culture dishes. At
the indicated times post infection, media were replaced by fresh
media, and virus titers in the culture fluids were determined by a
plaque assay on BHK-21 cells. Note that cells transfected with
VEEV/CSIN1 and VEEV/Cfrsh RNAs continued to release virus after 24
h post transfection, when VEEV TC-83 RNA transfected cells
developed a profound CPE. FIG. 9C shows survival of mice infected
with indicated viruses. Six-day-old NIH Swiss mice were inoculated
i.c. with indicated doses of viruses. Animals were monitored for
two months. No deaths occurred after day 9 post infection in any of
these experiments.
[0024] FIG. 10A-C shows downregulation of the importin-a/b-mediated
nuclear import in the cells infected with VEEV TC-83. BHK-21 cells
were either infected with packaged VEErep/4xTomato (FIG. 10A) or
VEErep/4xTomato-3.times.NLS (FIG. 10B) replicons at an MOI of
.about.20 inf.u/cell, or co-infected with VEErep/4xTomato-3xNLS
replicon and VEEV TC-83 at the same MOIs (FIG. 10C). Distribution
of 4xTomato and 4xTomato-3xNLS proteins was evaluated at 8 h post
infection. Panels: (a) distribution of the 4xTomato and
4xTomato-3xNLS proteins; (b) cell nuclei stained with SYTOX Green
(Invitrogen); (c) overlay of two images. Schematic representations
of used VEErep/4xTomato and VEErep/4xTomato-3xNLS replicons and
VEEV TC-83 genome are shown. Bars correspond to 20 mm.
[0025] FIG. 11A-11D shows the expression of C.sub.VEE affects
nuclear import of the proteins having different nuclear
localization signals. (FIG. 11A) BHK-21 cells were co-infected with
packaged VEErep/4xTomato-3xNLS and VEErep/C.sub.VEE/GFP. (a)
Distribution of 4xTomato-3xNLS protein; (b) distribution of GFP;
(c) overlay of two images. (FIG. 11B) BHK-21 cells were co-infected
with VEErep/4xTomato-M9 and VEErep/C.sub.VEE/GFP replicons. (a)
Distribution of 4xTomato-M9 protein; (b) distribution of GFP; (c)
overlay of two images. (FIG. 11C) BHK-21 cells were co-infected
with VEErep/4xTomato-H2b and VEErep/C.sub.VEE/GFP replicons. (a)
Distribution of 4xTomato-H2b protein; (b) distribution of GFP; (c)
overlay of two images. (FIG. 11D) Distribution of fluorescent
proteins in BHK-21 cells infected with either VEErep/C.sub.VEE/GFP,
or VEErep/4xTomato-M9, or VEErep/4xTomato-H2b. Cells infected with
VEErep/4xTomato-3xNLS are presented in FIG. 10. All of the images
were acquired at 8 h post infection. Bars correspond to 20 mm. The
schematic representation of the replicons is shown on each
panel.
[0026] FIG. 12 shows 5.times.10.sup.5 BHK-21 cells in 6-well Costar
plates were infected with the indicated replicons and viruses at an
MOI of 20 inf.u or PFU per cell, respectively. Proteins were
pulse-labeled with [.sup.35S]methionine at 8 h post infection and
analyzed on sodium dodecyl sulfate-10% polyacrylamide gel. Gel was
dried and autoradiographed. Positions of the expressed proteins of
interest are indicated at the right side of the gel.
[0027] FIG. 13A-13D shows an analysis of the 4xTomato-3xNLS
distribution in the cells, expressing either C.sub.SIN or different
variants of C.sub.VEE, fused with GFP. (FIG. 13A) BHK-21 cells were
co-infected with packaged VEErep/4xTomato-3xNLS and
VEErep/C.sub.SIN/GFP. (a) Distribution of 4xTomato-3xNLS protein;
(b) distribution of GFP; (c) overlay of two images. (FIG. 13B)
BHK-21 cells were co-infected with VEErep/4xTomato-3xNLS and
VEErep/C.sub.VEE1-68-GFP replicons. (a) Distribution of
4xTomato-3xNLS protein; (b) distribution of C.sub.VEE1-68-GFP; (c)
overlay of two images. (FIG. 13C) BHK-21 cells were co-infected
with VEErep/4xTomato-3xNLS and VEErep/C.sub.VEEfrsh-GFP replicons.
(a) Distribution of 4xTomato-3xNLS protein; (b) distribution of
C.sub.VEEfrsh-GFP; (c) overlay of two images. (FIG. 13D) BHK-21
cells were co-infected with VEErep/4xTomato-3xNLS and
VEErep/C.sub.VEE-GFP replicons. (a) Distribution of 4xTomato-3xNLS
protein; (b) distribution of C.sub.VEE-GFP; (c) overlay of two
images. All of the images were acquired at 8 h post infection. Bars
correspond to 20 mm. The schematic representation of the replicons
is shown on each panel.
[0028] FIG. 14A-14D show the distribution of VEEV nsP2 in the
cells, infected with VEEV TC-83. (FIG. 14A) nsP2 distribution in
BHK-21 cells, (FIG. 14B) in NIH 3T3 cells and (FIG. 14C) in HEK293
cells. Staining was performed at 8 h post infection. (FIG. 14D)
Staining of the mock-infected cells with VEEV nsP2-specific
antibodies. Panels (a) staining with VEEV nsP2-specific antibodies,
(b) nuclear staining with SYTOX Orange, (c) overlays of the images.
All of the images were acquired at 8 h post infection. Bars
correspond to 20 mm. The schematic representation of the replicons
is shown on each panel.
[0029] FIG. 15A-15C show that SINV nsP2 distribution depends on the
capsid protein, encoded by viral genome. (FIG. 15A) Intracellular
distribution of SINV nsP2 during SINV Toto 1101 replication or
(FIG. 15B) replication of SIN/VEEV recombinant virus in BHK-21
cells. (FIG. 15C) Staining of the mock-infected BHK-21 cells.
Panels (a) staining with the SINV nsP2-specific antibodies, (b)
nuclear staining with SYTOX Orange, (c) overlays of the images.
Staining was performed at 8 h post infection. Images were acquired
at 8 h post infection. Bars correspond to 20 mm. The schematic
representation of the replicons is shown on each panel. In the
schematic representations of viral genomes, SINV-specific sequences
are indicated by open boxes, VEEV sequences are indicated by filled
boxes.
[0030] FIG. 16 shows the VEEV nsP2-GFP fusion protein distribution
in the cells, infected with VEEV/nsP2GFP. BHK-21 cells were
infected with the chimeric virus at an MOI of 20 PFU/cell, and
nsP2/GFP distribution was evaluated at 8 h post infection on the
confocal microscope. Panel (a) distribution of VEEV nsP2/GFP, (b)
overlay of nsP2/GFP and nuclear staining with SYTOX Orange. Images
were acquired at 8 h post infection. Bars correspond to 20 mm. The
schematic representation of the viral genome is shown.
[0031] FIG. 17A-17B show the distribution of VEEV nsP2 in the cells
infected by the constructs encoding no C.sub.VEE. (FIG. 17A)
Distribution of VEEV nsP2 in the cells infected with VEErep/Cherry.
(a) Staining with VEEV nsP2-specific antibodies; (b) distribution
of Cherry; (c) overlay of two images. (FIG. 17B) Distribution of
VEEV nsP2-HA in the cells, infected with SINrep2V/nsP2VEE-HA
replicon. (a) Staining with anti-HA antibodies; (b) nuclear
staining with SYTOX Orange; (c) overlay of two images. All of the
images were acquired at 8 h post infection. Bars correspond to 20
mm. The schematic representation of the replicons is shown on each
panel.
[0032] FIG. 18A-18B show that C.sub.VEE does not interfere with
nuclear import in mosquito cells. C.sub.710 cells were transfected
by in vitro-synthesized VEErep/4xTomato-3xNLS and
VEErep/C.sub.VEE-GFP replicon RNAs (A), and VEErep/4xTomato-3xNLS
and VEErep/C.sub.VEE/GFP RNAs (B). Panels include (a) distribution
of 4xTomato-3xNLS protein; (b) distribution of C.sub.VEE-GFP and
GFP on (A) and (B), respectively; (c) overlay of two images. All of
the images were acquired at 24 h post transfection. Bars correspond
to 20 mm. The schematic representation of the replicons is shown on
each panel.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." Some
embodiments of the invention may consist of or consist essentially
of one or more elements, method steps, and/or methods of the
invention. It is contemplated that any method or composition
described herein can be implemented with respect to any other
method or composition described herein.
[0034] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0035] As used herein, the term "immunologically effective amount"
refers to an amount that results in an improvement or remediation
of the symptoms of the disease or condition due to induction of an
immune response. Those of skill in the art understand that the
effective amount may improve the patient's or subject's condition,
but may not be a complete cure of the disease and/or condition.
[0036] As used herein, "active immunization" is defined as the
administration of a immunogenic composition to stimulate the host
immune system to develop immunity against a specific pathogen or
toxin.
[0037] The immunogenic composition may comprise an adjuvant. As
used herein, "adjuvant" is defined as a substance which when
included in an immunogenic formulation non-specifically enhances
the immune response to an antigen.
[0038] The natural transmission cycle of the VEEV and other
alphaviruses implies their persistent replication in mosquito
vectors and development of high-titer viremia in vertebrate hosts
that is required for infection of new mosquitoes during the blood
meal. The level of viremia and its duration are the critical
factors that determine the successful continuation of the enzootic
cycle and virus persistence in nature. However, like any other
viral infection, alphavirus replication in vertebrate cells induces
a response aimed at downregulation of virus production and
activation of cell signaling, leading to activation of the
antiviral state in the uninfected cells and prevention of
successive rounds of infection. As did most, if not all of other
viruses, alphaviruses developed efficient mechanisms of
interference with the cellular response, and one of them is based
on the inhibition of cellular transcription (9, 13).
[0039] In the Old World alphaviruses SINV and SFV, the
nonstructural protein nsP2 is a key factor in the inhibition of
transcription of cellular messenger and ribosomal RNAs. However,
the New World alphaviruses VEEV and EEEV employ not the nsP2, but
the structural, capsid protein, for achieving the same goal. Capsid
protein is efficiently expressed in the infected cells and
distributed both in the cytoplasm and in the nuclei. Besides
packaging of viral genome RNA, VEEV- and EEEV-specific capsids are
transported into nuclei and cause global inhibition of cellular
transcription that strongly correlates with cytopathic effect
development. The results of this study demonstrate that both
cytopathic effect development and transcription inhibition are
determined by the same, short, N-terminal peptide of C.sub.VEE,
positioned between amino acid 33 and 69. It is difficult to expect
that such a short peptide has two separate functions:
transcriptional shutoff and cytopathic effect induction. Most
likely, cytopathic effect develops is a result of transcriptional
block. However, a possibility that both phenomena can be determined
by different mechanisms cannot be completely ruled out.
[0040] The identified functional peptide can be provisionally
divided into two domains: i) a previously defined a-helix, HI,
(amino acids 34-51) (31, 32) that is present in all of the
alphavirus capsids and has been shown to function in core assembly,
and ii) a downstream, highly positively charged peptide located
between amino acid 51 and 69. The amino acid sequences of both
domains demonstrate a strong conservation among the New World
alphaviruses, but these peptides differ from the sequences in the
Old World alphavirus capsid, which are nonfunctional in
transcription inhibition and incapable of causing CPE. Alterations
of each domain in C.sub.VEE34-68 have strong negative effects on
the ability of the entire C.sub.VEE or minimal C.sub.VEE30-68
peptide to cause transcriptional shutoff and CPE development.
However, these mutations appear to affect different functions.
C.sub.VEEGFP fusion lacking the HI domain (C.sub.VEED35-47GFP)
accumulated only in the cell nuclei, and C.sub.VEEfrsh having a
frame-shift mutation in the downstream peptide (aa 58-85) was no
longer transported to the nucleus. The latter frame-shift mutation
changed an amino acid 58-85 sequence in a very interesting way: the
peptide remained highly hydrophilic and became even more positively
charged. However, based on the computer prediction, the putative
NLSs, located between amino acids 64 and 85, were destroyed.
C.sub.VEEGFP fusion protein is larger than the proteins that can
passively diffuse to the nucleus, but a significant fraction of
this protein was found in that compartment. Since the frame-shift
mutation made fusion protein incapable of translocation to the
nuclei, this finding was a strong indication that the NLSs
predicted after amino acid 64 are indeed functional and drive the
C.sub.VEEGFP and, most likely, C.sub.VEE itself into the
nucleus.
[0041] The increase in nuclear localization of C.sub.VEE after HI
deletion might be explained in different ways. First, as described
for C.sub.SIN (31, 32), the HI deletion might strongly affect the
nucleocapsid assembly and, consequently, the balance between the
core-associated capsid cores and the free form of this protein
that, as indicated above is capable of translocation to the
nucleus. Another explanation is that in addition to the proposed
another peptide (50), functioning in C.sub.SIN binding to the
ribosomes, VEEV HI is involved in this function and, thus in
retaining of C.sub.VEE in the cytoplasm. Finally, C.sub.VEE-HI
might function as a nuclear export signal, and the balance between
C.sub.VEE import to the nucleus and its export determines its
intracellular distribution.
[0042] The most important result was, however, not in the changes
of C.sub.VEE compartmentalization due to HI deletion or mutations
in amino acids 58-85 peptide, but in the detected inability of the
mutated proteins to inhibit cellular transcription and cause
cytopathic effect. The accumulation of HI deletion mutant in the
nucleus did not noticeably affect cell biology and strongly
suggested that HI functions not only in the core assembly and
control of intracellular distribution of capsid; this sequence
appears to be also strongly involved in the development of
transcriptional shutoff. Similar conclusions can be made about the
C.sub.VEE52-68 domain: it certainly contains a functional NLS, but
the point mutations in 51 and 52 (outside the NLS) also made
C.sub.VEE incapable of inhibiting transcription. Moreover, the
addition of the artificial NLS to C.sub.VEE30-60GFP fusion (having
the natural NLS deleted) did not restore the ability of this
protein to inhibit cellular transcription, indicating that 52-68
peptide activities are likely more extensive than just acting as an
NLS. Thus, both domains, the HI and amino acids 52-68, appear to
have more sophisticated functions in regulation of cellular
transcription than control of capsid distribution alone.
[0043] One of the possible explanations for this new capsid
activity may lie in the modification of nucleocytoplasmic
transport. Significant fractions of C.sub.VEEGFP (FIGS. 8A-8B) and
C.sub.VEE30-68GFP were detected on the nuclear membrane, where they
demonstrated a distribution similar to that of the NPCs, suggesting
interaction of Nups and C.sub.VEE. To date, inhibition of nuclear
transport has been described only for a very limited number of
viruses, among which VSV, poliovirus, rhinovirus and cardiovirus
are better studied (16, 28, 33, 37). The VSV matrix protein
interacts with the nucleoporin Nup98 and export receptor Rae 1 (8).
Thus, M protein accumulates in the NPC (34), in which it
efficiently inhibits Rae 1-mediated mRNA nuclear export (34, 46)
and slows the rate of the nuclear import through importin
a/b1-dependent pathway (33). Interestingly, VSV also efficiently
inhibits cellular transcription (4), but the correlation between
inhibition of nucleocytoplasmic traffic and downregulation of
transcription has not been investigated. Picornaviruses have been
shown to alter nucleocytoplasmic transport either by the
protease-dependent processing of nucleoporins (17, 18) or by
disruption of the RanGDP/GTP gradient (37). Therefore, VEEV and,
most likely, other New World aphaviruses appear to join a growing
number of pathogens that interfere with the activation of cellular
genes that function in the antiviral response, by modifying
nucleocytoplasmic transport.
[0044] The importance of C.sub.VEE localization on the NPC is
currently supported by two findings: i) a significant fraction of
C.sub.SIN (the noncytopathic capsid) is present in the nucleus, but
is not associated with the nuclear membrane, and this might be a
plausible explanation of C.sub.SIN's inability to cause
transcriptional shutoff. ii) As indicated above, C.sub.VEE with a
deleted HI sequence is present in the nucleus in a high
concentration; however, it does not accumulate on the nuclear
membrane/nuclear pores, and this strongly correlates with
C.sub.VEED36-47 (having HI deleted) inability to inhibit
transcription. Moreover, it was observed that C.sub.VEE inhibits at
least one nuclear import pathway that is mediated by the
importin-a/b receptors, and prevents translocation of the SV40
NLS-containing proteins to the nuclei.
[0045] The importance of newly identified functions of C.sub.VEE
for virus replication was strongly supported by in vitro and in
vivo experiments with replicating VEEV TC-83, encoding modified
versions of capsid. The replacement of the entire N-terminal
fragment of C.sub.VEE with that of C.sub.SIN in VEEV/C.sub.sin1 did
not noticeably change replication of the virus in BHK-21 cells, and
the original VEEV TC-83 and VEEV/C.sub.sin1 demonstrated nearly
identical growth rates. However, the mutated virus was dramatically
less cytopathic and cells continued to release infectious virus
days after complete cytopathic effect development in VEEV
TC-83-infected samples. Moreover, this mutant was more attenuated
in vivo than the currently available vaccine strain TC-83. The
introduction of the above-described frame-shift mutations into the
capsid gene of TC-83 had a detectable negative effect on virus
replication. This lower level of virus replication might explain
its more attenuated phenotype in vivo. However, its strongly
altered ability to cause cytopathic effect in cultured cells points
to the possibility that a change in C.sub.VEE interactions with
cellular transcriptional machinery may also be involved.
[0046] Taken together, the present invention suggests new ways of
New World alphavirus attenuation: i) the identified critical domain
of VEEV, EEEV and WEEV capsid proteins can be modified by point
mutations or small deletions, or ii) the large fragments of the
protein can be replaced by the Old World alphavirus-derived
counterparts. The second direction may prove more promising,
because one of the distinguishing features of alphaviruses is in
their extraordinarily high mutation rates and adaptation.
Therefore, the effect of point mutations and small deletions, which
have a negative effect on virus growth rates are usually
neutralized by adaptive, compensatory mutations within a few
subsequent passages in vivo or in vitro. It will be, likely, more
difficult to adapt the entire N-terminal, SINV-specific fragment
(C1-98) or the entire C.sub.SIN, which did not become capable of
inducing transcriptional shutoff during the entire, previous virus
evolution.
[0047] In conclusion, the present invention developed attenuated
alphavirus that can be used as vaccines having irreversible,
attenuated phenotype. In general, representative alphaviruses of
the present invention have modified capsid proteins and are capable
of efficient replication in tissue culture but cannot cause disease
in the animals and immunized individuals. Specifically, the present
invention demonstrates that (i) the N-terminal fragments of VEEV,
EEEV and, most likely, of WEEV capsid proteins contain a
.about.35-aa-long peptide that functions in the inhibition of
cellular transcription and cytopathic effect development; (ii) the
identified, VEEV-specific peptide, C.sub.VEE30-68, includes two
domains with distinguished functions: the a-helix domain, HI, that
is critically involved in supporting the balance between the
presence of the protein in the cytoplasm and nucleus, and the
C-terminal peptide that contains the NLS(s). The integrity of both
domains determines the intracellular distribution of C.sub.VEE, and
both are essential for capsid function in the inhibition of
transcription; (iii) C.sub.VEE appears to interact with NPC, and
this interaction correlates with the protein's ability to cause
transcriptional shutoff, and, ultimately, cytopathic effect
development; and (iv) the replacement of the N-terminal fragment of
C.sub.VEE by its SINV-specific counterpart in VEEV TC-83 genome
does not affect virus replication in vitro, but makes it strongly
less cytopathic and more attenuated in vivo.
[0048] The present invention is directed to a method of attenuating
New World encephalitogenic alphavirus comprising: mutating one or
more than one amino acids in the amino terminal of the capsid
protein of the alphavirus; or replacing the entire capsid protein
or amino terminal of the capsid protein of the alphavirus by capsid
protein or amino terminal of less pathogenic Old World alphavirus.
Examples of the mutation is not limited to but may include a point
mutation of the amino acids in the amino terminal of the capsid
protein. The amino acid(s) mutated or replaced is not limited to
but may include amino acids 33-68 of Venezuelan Equine Encephalitis
virus capsid protein, amino acids 36-72 of Eastern Equine
Encephalitis virus capsid protein or amino acids 36-72 of Western
Equine Encephalitis virus capsid protein. Furthermore, the Old
World alphavirus may include but is not limited to Sindbis, Semliki
Forest, Ross River, Aura and other antigenically related viruses.
Additionally, the attenuated New World, encephalitogenic alphavirus
may be capable of replicating in vitro but cannot cause disease in
animals and in immunized individuals.
[0049] The present invention is also directed to an immunogenic
composition, comprising the attenuated New World encephalitogenic
alphavirus generated by the method described supra. Such attenuated
New World encephalitogenic alphavirus may comprise mutations of one
or more than one amino acids in the amino terminal of the capsid
protein of the alphavirus, deletion of entire capsid protein or
deletion of amino terminal of the capsid protein. The deleted
capsid protein or amino terminal of the capsid protein may be
replaced by capsid protein or amino terminal of the capsid protein
of less pathogenic Old World alphavirus. Examples of the amino
acid(s) mutated or replaced may include but is not limited to amino
acids 33-68 of Venezuelan Equine Encephalitis virus capsid protein,
amino acids 36-72 of Eastern Equine Encephalitis virus capsid
protein or amino acids 36-72 of Western Equine Encephalitis virus
capsid protein. Examples of the Old World alphavirus may include
but is not limited to Sindbis, Semliki Forest, Ross River, Aura and
other antigenically related viruses.
[0050] The present invention is also directed to a method of
preventing an infection caused by Old and New World
encephalitogenic alphavirus in a subject, comprising: administering
an immunologically effective amount of the immunogenic composition
described supra to the subject. Examples of the New World
encephalitogenic alphavirus may include but is not limited to
Venezuelan Equine Encephalitis virus, Western Equine Encephalitis
virus or Western Equine Encephalitis virus and those of the Old
World alphavirus may include but is not limited to Sindbis virus,
Semliki Forest virus, Ross River virus or Aura virus. Such an
immunogenic composition may be administered subcutaneously or
intramuscularly. Additionally, the subject may be a human or an
animal, where the subject may be a healthy subject or a subject who
is likely to be exposed to the alphavirus.
[0051] Treatment methods will involve preventing an infection in a
subject with an immunologically effective amount of a composition
described supra. An immunologically effective amount is described,
generally, as that amount sufficient to detectably and repeatedly
induce an immune response so as to prevent, ameliorate, reduce,
minimize or limit the extent of a disease or its symptoms. More
specifically, it is envisioned that the treatment with the
immunogenic composition elicits an antibody response and/or
decreases the viral load in the subject to prevent the infection
caused by the New World and Old World encephalitogenic
alphavirus.
[0052] The immunologically effective amount of the immunogenic
composition to be used are those amounts effective to produce
beneficial results, particularly with respect to preventing the
infection caused by the New World and Old World encephalitogenic
alphavirus, in the recipient animal or human. Such amounts may be
initially determined by reviewing the published literature, by
conducting in vitro tests or by conducting metabolic studies in
healthy experimental animals. Before use in a clinical setting, it
may be beneficial to conduct confirmatory studies in an animal
model, preferably a widely accepted animal model of the particular
disease to be treated. Preferred animal models for use in certain
embodiments are rodent models, which are preferred because they are
economical to use and, particularly, because the results gained are
widely accepted as predictive of clinical value.
[0053] The immunogenic composition disclosed herein may be
administered either alone or in combination with another drug or a
compound. Such a drug or compound may be administered concurrently
or sequentially with the immunogenic composition disclosed herein.
The effect of co-administration with the immunogenic composition is
to lower the dosage of the drug or the compound normally required
that is known to have at least a minimal pharmacological or
therapeutic effect against the disease that is being treated.
Concomitantly, toxicity of the drug or the compound to normal
cells, tissues and organs is reduced without reducing,
ameliorating, eliminating or otherwise interfering with any
cytotoxic, cytostatic, apoptotic or other killing or inhibitory
therapeutic effect of the drug or the compound.
[0054] The composition described herein and the drug or the
compound may be administered independently, either systemically or
locally, by any method standard in the art, for example,
subcutaneously, intravenously, parenterally, intraperitoneally,
intradermally, intramuscularly, topically, enterally, rectally,
nasally, buccally, vaginally or by inhalation spray, by drug pump
or contained within transdermal patch or an implant. Dosage
formulations of the composition described herein may comprise
conventional non-toxic, physiologically or pharmaceutically
acceptable carriers or vehicles suitable for the method of
administration.
[0055] The immunogenic composition described herein and the drug or
the compound may be administered independently one or more times to
achieve, maintain or improve upon a therapeutic effect. It is well
within the skill of an artisan to determine dosage or whether a
suitable dosage of either or both of the immunogenic composition
and the drug or the compound comprises a single administered dose
or multiple administered doses.
[0056] As is well known in the art, a specific dose level of such
an immunogenic composition generated thereof for any particular
patient depends upon a variety of factors including the activity of
the specific compound employed, the age, body weight, general
health, sex, diet, time of administration, route of administration,
rate of excretion, drug combination, and the severity of the
particular disease undergoing therapy. The person responsible for
administration will determine the appropriate dose for the
individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics
standards.
[0057] One of skill in the art realizes that the immunologically
effective amount of the immunogenic composition generated thereof
can be the amount that is required to achieve the desired result:
enhance antibody response, decrease the bacterial load, etc.
[0058] Administration of the immunogenic composition of the present
invention to a patient or subject will follow general protocols for
the administration of therapies used in treatment of infections
taking into account the toxicity, if any, of the components in the
immunogenic composition, the antibody and/or, in embodiments of
combination therapy, the toxicity of the antibiotic. It is expected
that the treatment cycles would be repeated as necessary. It also
is contemplated that various standard therapies, as well as
surgical intervention, may be applied in combination with the
described therapy.
[0059] As is known to one of skill in the art the immunogenic
composition described herein may be administered along with any of
the known pharmacologically acceptable carriers. Additionally the
immunogenic composition can be administered via any of the known
routes of administration such as subcutaneous, intranasal or
mucosal. Furthermore, the dosage of the composition to be
administered can be determined by performing experiments as is
known to one of skill in the art.
[0060] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion. One skilled in the
art will appreciate readily that the present invention is well
adapted to carry out the objects and obtain the ends and advantages
mentioned, as well as those objects, ends and advantages inherent
herein. Changes therein and other uses which are encompassed within
the spirit of the invention as defined by the scope of the claims
will occur to those skilled in the art.
Example 1
Cell Cultures
[0061] BHK-21 cells were provided by Dr. Sondra Schlesinger
(Washington University, St. Louis, Mo.). NIH 3T3 cells were
obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). Both cell lines were maintained at 37.degree. C.
in alpha minimum essential medium (aMEM) supplemented with 10%
fetal bovine serum (FBS) and vitamins.
Example 2
Plasmid Constructs
[0062] VEErepL replicon, having a mutation in the nsP2 gene,
Q739.fwdarw.L, is described elsewhere (12, 35). The distinguishing
feature of this replicon is in its noncytopathic phenotype and low
level of genome RNA replication and transcription of the subgenomic
RNA. Such replicons do not overproduce the proteins of interest and
generate biologically relevant data. VEErepL/GFP/Pac replicon, used
as a noncytopathic control in many experiments, is described
elsewhere (11). The genes of tested proteins were cloned into
VEErepL under the control of the subgenomic promoter, and the
second promoter was driving the expression of puromycin acetyl
transferase (Pac) that makes the replicon-containing cells
resistant to translational arrest caused by puromycin. All of the
tested cassettes expressing capsid protein with different deletions
were synthesized by PCR and sequenced before cloning into the
vector replicon as GFP fusions. The selection of frame-shift mutant
of CVEE is described elsewhere (12), and the corresponding gene was
synthesized by RT-PCR using the RNA, isolated from the growing,
replicon-containing cells. The capsid-specific peptides were
separated from GFP by short, flexible peptide 4xGly. All of the GFP
fusions were designed in a way to avoid cleavage by the
capsid-encoded protease. To achieve this, the last amino acid in
capsid was deleted. An additional AUG codon was added to all of
cassettes encoding VEEV- and EEEV-specific peptides that did not
contain the initiating AUG. VEEV/CSIN1 encoded the infectious VEEV
TC-83 genome, in which the sequence encoding amino acid 1-110 of
CVEE was replaced by sequence encoding aa 1-1-98 of CSINV.
VEEV/CVEEfrsh genome encoded capsid, having previously identified
frame shift mutations (12).
Example 3
RNA Transcriptions
[0063] Plasmids were purified by centrifugation in CsCl gradients.
Before being subjected to a transcription reaction, plasmids were
linearized using the MluI or NotI restriction sites located
downstream of the poly(A) sequence of VEE replicons. RNAs were
synthesized by SP6 RNA polymerase in the presence of a cap analog
by described conditions (38). The yield and integrity of
transcripts were analyzed by gel electrophoresis under
non-denaturing conditions. RNA concentration was measured on a
Fluor Chem imager (Alpha Innotech), and transcription reactions
were used for electroporation without additional purification.
Example 4
Analysis of Cytotoxicity of the Constructs
[0064] BHK-21 cells were electroporated as described (29). In all
of the experiments, 5 mg of the in vitro-synthesized RNAs was used
per electroporation of 5.times.10.sup.6 cells. Next, the aliquots
of the cells were seeded into 6-well Costar plates for analysis of
cell proliferation and viability as described elsewhere (11, 12).
Puromycin selection (10 mg/ml) was performed between 6 and 48 h
post transfection. Then cells were incubated in puromycin-free
media, and viable cells were counted at the times indicated in the
figures. In parallel, different dilutions of the electroporated
cells were seeded into 100-mm tissue culture dishes. At 6 h post
transfection, puromycin was added to the media to a concentration
of 10 mg/ml. Colonies of Pur.sup.R cells were stained with crystal
violet at days 4-9 post transfection, depending on their growth
rates. The results are expressed in colony-forming units (CFU) per
mg of RNA used for transfection.
Example 5
Analysis of Cellular Transcription
[0065] BHK-21 cells were electroporated by 5 mg of the in
vitro-synthesized RNAs and one-tenth of the cells were seeded into
35-mm culture dishes. At 6 h post transfection, puromycin was added
to the media to a concentration of 10 mg/ml. At indicated times
post electroporation, the cellular RNAs were labeled for the time
periods given in figure legends in the complete aMEM supplemented
with 10% FBS and 20 mCi/ml [.sup.3H]uridine without addition of
ActD. The RNAs were isolated by TRizol reagent as recommended by
the manufacturer (Invitrogen) and analyzed by agarose gel
electrophoresis as previously described (5).
[0066] To assess the total RNA synthesis, the RNA samples on the
Whatman 3MM paper were washed with cold 10% Trichloroacetic acid
(TCA), and radioactivity was measured by liquid scintillation
counting and normalized on the number of viable cells determined by
the above described tests.
Example 6
Infectious Center Assay
[0067] Five mg of in vitro-synthesized, full-length RNA transcripts
of viral genomes was used per electroporation. Ten-fold dilutions
of electroporated BHK-21 cells were seeded in six-well Costar
plates containing subconfluent naive cells. After 1 h incubation at
37.degree. C. in a 5% CO.sub.2 incubator, cells were overlaid with
2 ml of MEM-containing 0.5% Ultra-Pure agarose supplemented with 3%
FBS. Plaques were stained with crystal violet after 2 days
incubation at 37.degree. C.
Example 7
Viral Replication Analysis
[0068] To exclude any effect of possible virus evolution on the
replication efficiency, virus growth rates were evaluated directly
after electroporation of the in vitro-synthesized RNA into the
cells. BHK-21 cells were electroporated by 5 mg of the RNAs, and
one-fifth of the cells were seeded into 35-mm culture dishes. At
the indicated times post infection, media were replaced by fresh
media, and virus titers in the culture fluids were determined by a
plaque assay on BHK-21 cells as previously described (26).
Example 8
Immunization and Challenge with Virulent VEEV
[0069] Weanling, female, six-day-old mice were inoculated i.c. with
VEE TC-83 or other designed viruses at a dose of 10.sup.7
plaque-forming units in a total volume of 20 .mu.l of PBS. After
vaccination, each cohort of 10 animals was maintained for 21 days
without any manipulation. Mice were observed twice daily for
clinical illness (noting those with ruffled coat, depression,
anorexia and/or paralysis) and/or death.
Example 9
Microscopy
[0070] BHK-21 cells were seeded on glass chamber slides (Nunc) and
transfected with 2 mg of in vitro-synthesized replicon RNA using
Lipofectamine 2000 according to the manufacturer's instructions
(Invitrogen). Then, at 12 h post transfection, they were fixed in
3% formaldehyde in phosphate buffered saline (PBS), and the
distribution of the GFP-containing fusion proteins was analyzed on
a Zeiss LSM510 META confocal microscope using a 63X 1.4NA oil
immersion planapochromal lens. For staining of nucleopore complexes
(NPC), cells were additionally permeablized with 0.5% Triton X-100,
stained with MAb414 antibodies (Covance Innovative Antibodies) and
AlexaFluor 546-labeled secondary antibodies and analyzed using a
confocal microscope.
Example 10
The Mutations in VEEV Capsid Protein Affect its Ability to Cause
Both CPE and Transcription Inhibition
[0071] VEEV capsid was expressed from VEEV replicons having a
mutation in nsP2 that decreased RNA replication as described (12).
Capsid production caused rapid CPE development and inhibited RNA
polymerase I- and II-dependent cellular transcription. However,
fewer than 0.1% of the cells continued to grow, and their ability
for growth correlated with an accumulation of mutations in the
replicon-encoded capsid gene. The majority of mutations destroyed
the ORF downstream of the amino acid 50 of the capsid-coding
sequence, but others changed either a single amino acid:
K.sub.51.fwdarw.E or Q.sub.52.fwdarw.P, or a short peptide between
amino acid 57 and 86 (58-85-frame shift). However, the latter point
and frame-shift mutations were detected only in the capsid gene
(mutC.sub.VEE) that encoded the protein with inactive protease
(S.sub.226.fwdarw.A mutant). Therefore, it was not clear whether
the K.sub.51.fwdarw.E, Q.sub.52.fwdarw.P and frame shift mutations
strongly affected the ability of the protein to cause CPE, or if
this was a synergistic effect of these mutations and inactivation
of protease activity by the S.sub.226.fwdarw.A replacement.
[0072] To distinguish between these two possibilities and to
further evaluate the effect of the mutations on C.sub.VEE function,
the above described K.sub.51.fwdarw.E, Q.sub.52.fwdarw.P and
frame-shift mutations were cloned into VEErepUmutC.sub.VEE/Pac
replicon, in which capsid gene encoded a protease mutant, and into
similar replicon that encoded a wt capsid, VEErepUC.sub.VEE/Pac
(FIGS. 1A-1B). All of the replicons were synthesized in vitro, and
equal amounts of RNAs were transfected into BHK-21 cells. Replicons
encoding GFP and capsids without the indicated mutations were used
as controls. The cytopathicity of the expressed capsids was
examined by evaluating the number of Pur.sup.R foci formed per mg
of transfected RNA (FIGS. 1A-1B) and measuring the growth of the
Pur.sup.R cells (FIGS. 1C-1D). Additionally, the ability of the
expressed proteins to inhibit cellular transcription was also
assessed (FIGS. 1E-1F).
[0073] All of the mutations previously identified in the
mutC.sub.VEE gene (K.sub.51.fwdarw.E, Q.sub.52.fwdarw.P and
58-85-frame shift) made the capsid and designed replicons
noncytopathic, regardless of their presence in C.sub.VEE or
mutC.sub.VEE. These mutations also made capsids incapable of
inhibiting cellular transcription. In repeated experiments, cells
containing replicons driving the expression of the indicated capsid
mutants demonstrated the same growth rates as untransfected control
or cells containing VEErepL/GFP/Pac. These data unambiguously
demonstrated that C.sub.VEE-encoded protease activity is not
involved in CPE development and downregulation of cellular
transcription, but these phenomena were, most likely, the function
of the N-terminal capsid-specific peptide (amino acid 1-110) that
was previously identified as a domain functioning in RNA packaging
and nucleocapsid formation.
Example 11
Deletion Analysis of the VEEV Capsid Protein
[0074] Alphavirus capsid protein was shown to contain two
structural domains. The C-terminal domain expresses a protease
activity required for co-translational self-cleavage of capsid from
the structural polyprotein (19, 20). The N-terminal domain is
highly positively charged (39) and functions in packaging of the
viral genome during core assembly (42). This domain is known to be
unfolded (7), except for a short peptide that is predicted to form
an alpha helix (HI) and is present in the capsid of all
alphaviruses (31, 32). This data indicated that the N-terminal, and
not the C-terminal domain determines C.sub.VEE function in CPE
development and inhibition of cellular transcription. Therefore, to
identify a particular peptide that exhibits these inhibitory
effects, a detailed deletion analysis was preformed wherein
N-terminal C.sub.VEE fragments were expressed as GFP fusions from
the VEErepL replicon. The use of GFP-tagged fragments allowed
following changes in the intracellular distribution of the protein
and to some extent, mimic the natural structure of original
C.sub.VEE, because in this case, the folded, C-terminal domain was
replaced by GFP that also demonstrates a globular structure. Such
fusion proteins had molecular weights similar to that of the
C.sub.VEE.
[0075] To make the data consistent with the previous results, the
fusions were expressed from the VEErepL replicon. Both the
C.sub.VEE 110 GFP fusion that expressed the entire N-terminal
domain, and the C.sub.VEE80GFP (FIG. 2A), having the highly
positively charged fragment deleted, were highly efficient in
causing both cell death (FIG. 2B) and inhibition of cellular
transcription (FIG. 2C), and very few replicon-containing cells
developed Pur.sup.R foci. These data indicated that the critical
peptide was located upstream of the deletions made.
[0076] The next set of deletions was designed based on the
alignment of the N-terminal C.sub.VEE fragment with that of other
alphaviruses. The high number of amino acids in the 34-68 peptide
of C.sub.VEE is identical to those in the corresponding fragments
of other New World alphaviruses, EEEV and WEEV, but not in the Old
World alphaviruses (whose capsids are not cytopathic) (FIG. 3A).
This finding pointed to the possibility that these amino acids
might determine the activity of the New World alphavirus capsids in
transcription inhibition. Therefore, the sequence of the
C.sub.VEE68 peptide encoding the entire conserved peptide was fused
with GFP and expressed it from VEErepL replicon (FIG. 3B). Other
constructs had either partial deletion of this sequence
(C.sub.VEE60GFP) or the deletion of the entire conserved fragment
downstream of the amino acid 33 (C.sub.VEE33GFP). The results were
in agreement with the assumption about the critical role of the
amino acid 34-68 peptide in virus-host cell interactions: the
expression of C.sub.VEE68GFP fusion caused CPE (FIG. 3C) and, by 24
h post transfection, downregulated cellular transcription to an
almost undetectable level (FIG. 3D). C.sub.VEE60 and C.sub.VEE34
were incapable of causing transcriptional shutoff, and GFP-tagged
peptides were as non-cytopathic as GFP itself. Taken together,
these data indicated that a protein fragment located upstream of
the amino acid 68 is critically involved in the inhibition of
cellular transcription and cytopathic effect development.
[0077] To define the beginning of the critical peptide, the
C.sub.VEE deletion mutants were designed in the context of complete
C.sub.VEEGFP fusion. Therefore, initial experiments were aimed at
demonstrating that such fusion has exactly the same functions in
modification of cell biology as C.sub.VEE itself. The last amino
acid in the capsid was deleted to avoid cleavage of the fusion by
capsid-associated protease, and fusion with GFP was performed
through a flexible peptide. The in vitrosynthesized
VEErepL/C.sub.VEEGFP/Pac and control replicons VEErepUC.sub.VEE/Pac
and VEErepL/GFP/Pac (FIG. 4A) were transfected into BHK-21 cells.
C.sub.VEEGFP-expressing cells developed cytopathic effect at
exactly the same rate as those expressing C.sub.VEE (FIG. 4B) and
demonstrated the same inhibition of cellular transcription (FIG.
4C). The mutation introduced into the C-terminus abolished
cleavage, and the detected phenotype could not be explained by
partial processing (FIG. 4D).
[0078] Further, the fragment encoding the peptide upstream of the
HI (aa 2-31), the upstream fragment and a part of the HI (amino
acids 2-40) and the HI only (amino acids 35-47) were deleted (FIG.
5A). The first deletion did not abolish the ability of the fusion
protein to cause a cytopathic effect; however, the deletions of
amino acids 2-40 and 35-47 made fusion proteins completely
incapable of both CPE induction (FIG. 5B) and inhibiting cellular
transcription. Thus, the results strongly indicated that the entire
HI is required for the capsid to be active in both processes.
[0079] To further confirm the conclusion that the C.sub.VEE34-68
peptide can function as the entire capsid in causing CPE cellular
transcription inhibition, cassette wherein the GFP gene was fused
with this minimal C.sub.VEE30-68-coding sequence was designed. Four
amino acids (amino acids 30-33) were left upstream of the HI to
preserve its proper folding. An additional AUG codon was cloned
upstream of the C.sub.VEE sequence, and fusion with GFP was
performed through a flexible linker. VEErepL/C.sub.VEE30-68GFP/Pac
replicon was as cytopathic as VEErepLUC.sub.VEEGFP/Pac (FIGS.
6A-6B), and the replicon expressing a shorter, C.sub.VEE30-60,
version of the protein did not noticeably affect cell growth. This
finding could be possibly explained by the fact that the
C.sub.VEE30-60 peptide had the putative, computer-predicted NLS (aa
64-68) deleted, leading to a less efficient translocation of the
protein to the nucleus. To additionally study this possibility, a
standard 3xNLS was cloned into the C-terminus of the GFP of all
three constructs. These NLSs did not have any effect on
C.sub.VEE30-68GFP and C.sub.VEEGFP functioning, and did not improve
the ability of C.sub.VEE30-60GFP to inhibit cellular
transcription.
[0080] Taken together, the results of this study strongly indicated
that the peptide, located between amino acids 33 and 69 of
C.sub.VEE, is critically involved in CPE development and
transcription inhibition. Both the Hi peptide and the downstream
sequence (amino acids 52-68) are required for capsid function. The
HI deletion and modifications of the downstream peptide make
C.sub.VEE30-68 nonfunctional. Moreover, the amino acids 52-68
peptide appeared to function not only as NLS, but is likely
directly involved in the development of transcriptional
shutoff.
Example 12
The EEEV-Specific, HI-Containing Peptide Functions Similar to its
VEEV-Specific Counterpart
[0081] As described supra, the C.sub.VEE30-68 peptide demonstrates
a significant level of conservation among the New World
alphaviruses (FIG. 3A). Therefore, it is reasonable to expect that
it has similar mode of functioning in EEEV and WEEV. To test this
possibility, amino acids 33-71 of C.sub.EEE (which corresponds to
aa 30-68 of C.sub.VEE capsid) was expressed as a GFP fusion from
VEErepL replicon (FIG. 7A). Since C.sub.EEE was not studied as
intensively as C.sub.VEE, whether the determinant of its function
in transcription inhibition and the NLS are located in the same
peptide (as they are in C.sub.VEE) was not known. By this reason,
an additional 3xNLS was cloned into the C terminus of GFP of
C.sub.EEE33-71GFP fusion. After transfection of the in
vitro-synthesized RNAs, cells expressing C.sub.EEE33-71GFP.sub.NLS
fusion demonstrated the same rates of CPE development as did those
with C.sub.VEE30-68GFP.sub.NLS (FIG. 7B), and transcription in
these cells was inhibited to the same level (FIG. 7C). This result
strongly indicated that the ability to interfere with the
transcription of cellular RNAs is a feature of the same peptide in
capsid proteins derived from the New World alphaviruses, and this
group of viruses appears to employ similar mechanisms of
interference with cellular gene expression.
Example 13
Intracellular Distribution of VEEV Capsid
[0082] As demonstrated previously, C.sub.VEE is distributed not
only in the cytoplasm, but also in the nuclei of virus-infected
cells (12). Its presence in the nucleus might be determined by a
combination of active nuclear import and passive diffusion. Based
on computer predictions, this protein appears to contain a number
of NLS-like sequences that might promote its transport into the
nucleus, and the majority of these putative signals is concentrated
within the peptide between amino acids 57-86, which was changed by
two attenuating, frame-shift mutations (12)
(VEErepL/C.sub.VEEfrsh/Pac in FIG. 1B). These data indicated that
the putative NLS(s) might be functional and important for C.sub.VEE
activity in the inhibition of transcription. Additionally to
investigate this, the intracellular distribution of C.sub.VEEGFP
and C.sub.VEEfrshGFP fusions expressed by VEErepL replicons was
compared. These fusion proteins are too large for passive diffusion
through the nuclear pores (30), and their presence in the nucleus
should be determined by NLS-dependent, active transport. The
results presented in FIG. 8A demonstrate that a significant
fraction of C.sub.VEEGFP was transported into the nucleus, and the
amino acids 58-85-frame-shift mutation blocked the ability of the
protein to accumulate in the nucleus and on the nuclear
membrane.
[0083] The presence of active NLS(s) in the capsid protein raised
the issue of whether this protein does not completely concentrate
in the nucleus. Its presence in the cytoplasm might certainly be
explained by binding to the ribosomes and/or core assembly.
However, the study of the intracellular distribution of another
C.sub.VEEGFP mutant suggested an alternative explanation.
C.sub.VEEGFP fusion that had an Hi deleted
(VEErepL/C.sub.VEED35-47GFP/Pac replicon in FIG. 5A) accumulated
only in the nuclei of the transfected cells (FIG. 8A). This was an
indication that the HI is either i) required for binding of capsid
to cytoplasmic organelles, ii) it stimulates an immediate assembly
of capsid molecules into higher-order, core structures, or iii) it
functions as a nuclear export signal supporting the balance between
the nuclear and cytoplasmic capsid concentrations.
[0084] The more detailed analysis of C.sub.VEEGFP
compartmentalization revealed another interesting feature of this
protein. Significant fractions of C.sub.VEEGFP (FIG. 8B) and
C.sub.VEE30-68GFP fusions were detected in the nuclear rim, where
both proteins demonstrated a punctuated pattern of distribution
reminiscent of that of the nuclear pore complexes (NPC). Cells
expressing this fusion protein from VEErepL replicon were stained
with nuclear pore-specific antibody (Mab414), which recognizes the
conserved FxFG repeats in several nucleoporins, namely Nup62,
Nup98, Nup153, Nup214 and Nup358. By using confocal microscopy,
co-localization of C.sub.VEEGFP was demonstrated with the Mab414
stained NPCs. In addition, some large, C.sub.VEEGFP-containing
complexes that were also capable of Mab414-binding were detected in
the cytoplasm. It is speculated that these structures are the
annulate lamellae (22) that were shown to contain nuclear pore-like
complexes. Thus, the results presented herein indicate that the wt
capsid and its C.sub.VEE30-68 variant are capable of binding to
NPC. Alterations of HI or replacement of the HI-containing fragment
by that of SINV capsid abolished localization of fusion proteins on
the nuclear membrane.
Example 14
Attenuated VEEV Variants
[0085] As we indicated above, C.sub.VEE expression by VEErepL
determined the development of CPE and transcriptional shutoff in
cells of vertebrate origin. However, the capsid protein of another
alphavirus, SINV, was incapable of causing both phenomena. These
results suggested that VEEV might be attenuated by making the
extended mutations in the critical C34-68 amino acid peptide or by
replacing this peptide with the corresponding C.sub.SIN-derived
fragment. Two possible variants tested herein (FIG. 9A) were
designed on the basis of the VEEV TC-83 genome. The original TC-83
strain is attenuated for adult mice, but is lethal for weanling
mice after i.c. inoculation. Therefore, this virus can be used for
making additional genetic manipulations and analysis of their
effect on pathogenesis. In VEEV/C.sub.SIN1, the natural VEEV capsid
was replaced by a chimeric version, in which the entire N-terminal
fragment, located upstream of the protease domain (aa 1-110), was
replaced by its SINV-specific counterpart (amino acids 1-98). The
second variant, VEEV/Cfrsh, encoded the capsid with the above
described frame-shift mutations that changed the peptide between
amino acid 57 and 86. It should be noted that the indicated frame
shift did not change the highly hydrophilic nature of the peptide
and even increased the number of positively charged amino acid in
the N-terminus. However, as described supra, the frame shift
affected the putative NLS. Both designed, in vitro-synthesized
genomes were as infectious as a control, VEEV TC-83 RNA and
generated homogeneous plaques in the infectious center assay, a
finding indicating that no additional mutations were required for
virus viability. In BHK-21 cells, both viruses demonstrated growth
rates comparable to those of VEEV TC-83 (FIG. 9B); however, in
contrast to TC-83, they became noncytopathic, and did not stop cell
growth, while infected cells were incubated in the media
supplemented with 10% FBS. Nevertheless, they were still capable of
forming plaques in BHK-21 cells under agarose cover having low
serum concentration.
[0086] Harvested viruses were further tested for their
pathogenicity in suckling mice. Animals were i.c. inoculated with
10.sup.7 PFU of VEEV/C.sub.SIN1, VEEV/Cfrsh and two indicated doses
of VEEV TC-83 (FIG. 9C). Both doses of the latter virus were
universally lethal for mice of this age. VEEV/C.sub.SIN1, and
VEEV/Cfrsh caused death only in part of mice, in spite of our
having used very high doses. Thus, modification of C.sub.VEE by
replacing the amino terminal fragment with the SINV-specific
counterpart or by a frame-shift mutation, affecting the NLS, caused
an additional attenuation of the VEEV TC-83. These data
demonstrated the importance of C.sub.VEE and the identified peptide
C.sub.VEE34-68, in particular, for virus replication and
pathogenesis.
[0087] The present invention also investigated the effect of
C.sub.VEE on nucleocytoplasmic transport and demonstrated that i)
both VEEV capsid and its active peptide (aa 33-68), block multiple
nuclear import pathways, but do not noticeably affect the passive
diffusion of small proteins from the cytoplasm to the nucleus. ii)
This activity is specific for C.sub.VEE, but not for the capsid
derived from the Old World alphavirus, Sindbis virus (SINV). iii)
C.sub.VEE expression does not noticeably affect nuclear import in
the cells of mosquito origin. This inability to affect nuclear
traffic provides one of the plausible explanations for lack of
profound cytopathic effect (CPE) in the VEEV-infected arthropod
cells and persistent, life-long replication of VEEV in the mosquito
vectors without noticeable effect on their biological
functions.
Example 15
Cell Cultures
[0088] BHK-21 cells were obtained from Dr. Paul Olivo (Washington
University, St. Louis, Mo.). NIH 3T3 cells were obtained from the
American Type Tissue Culture Collection (Manassas, Va.). HEK293
cells were provided by Dr. Robert Davey (University of Texas
Medical Branch at Galveston). These cell lines were maintained at
37.degree. C. in alpha minimum essential medium (aMEM) supplemented
with 10% fetal bovine serum (FBS) and vitamins. Mosquito C.sub.710
cells were obtained from Dr. Henry Huang (Washington University,
St. Louis, Mo.). They were propagated in DMEM supplemented with 10%
heat-inactivated FBS and 10% tryptose phosphate broth (TPB).
Plasmid Constructs
[0089] pVEErep/4xTomato plasmid encoded the VEEV replicon, in which
two tdTomato gene were fused in frame through a short linker
GHGTGSGGSGSS (SEQ ID NO: 5), which was previously applied for
tandem cloning of other fluorescent proteins, and the entire
cassette was cloned under control of the subgenomic promoter of the
replicon. The tdTomato encodes a dimer of modified DsRed, and,
thus, the entire cassette was designated a 4xTomato to emphasize
that this is a tetrameric protein. Further modifications of this
expression cassette, which were aimed at fusing the 4xTomato with
different nuclear localization signals (NLSs), were made by using
PCR or by cloning the sequences that were designed from the
oligonucleotides. pVEErep/C.sub.VEE/GFP encoded VEEV replicon, in
which one of the subgenomic promoters controlled the expression of
VEEV capsid, and the second promoter drove GFP expression. The
expression of this marker was used for demonstrating the presence
of the replicon in the cells. pVEErep/C.sub.SIN/GFP had the same
design, but encoded a SINV capsid protein (C.sub.SIN) under the
control of the subgenomic promoter. pVEErep/C.sub.VEE-GFP contained
a VEEV replicon having only one subgenomic promoter that drove the
expression of the C.sub.VEE-GFP fusion protein. To avoid cleavage,
the last aa in C.sub.VEE was deleted, and fusion was performed
through the short linker peptide (Gly).sub.4.
pVEErep/C.sub.VEE1-68-GFP and pVEErep/C.sub.VEEfrsh-GFP had a
similar design, but encoded in the fusion protein only the aa 1-68
of VEEV capsid or C.sub.VEE with previously identified, frame-shift
mutations (18), affecting aa 58-85. The latter frame-shift
mutations made the capsid incapable of causing transcriptional
shutoff and CPE development. pSINrep2V/Ubi-nsP2.sub.VEE-HA encoded
a SINV replicon that had a mutation in the nsP2/nsP3 cleavage site,
and the subgenomic promoter controlled the expression VEEV nsP2,
tagged with HA sequence. This replicon was incapable of P23
processing, and, thus, the unprocessed SINV nsP2 did not interfere
with VEEV nsP2 transport into the nucleus. Ubi was used to promote
expression of VEEV nsP2 with natural Ala at the amino terminus.
pVEErep/Cherry contained a VEEV replicon that encoded one of the
red fluorescent proteins, Cherry, under the control of the
subgenomic promoter. All of the above-described, VEEV and SINV
replicon-based cassettes were synthesized by PCR-based techniques,
and all of heterologous genes were sequenced. All of the constructs
that we used are presented in the corresponding figures. Sequences
of the recombinant plasmids can be provided upon request. VEEV and
SINV helpers used for replicons' packaging.
Viruses
[0090] Vaccine strain VEEV TC-83 and SINV Toto1101 were rescued
from the infectious cDNA clones. VEEV/nsP2GFP virus was developed
by random GFP insertion mutagenesis using the same strategy as
described for SINV. The GFP insertion was made after aa 3 of nsP2
gene in VEEV TC-83 genome, and, in contrast to similar SINV
constructs, this additional sequence had only a minor effect on the
efficiency of P123 cleavage. SIN/VEEV chimeric virus encoded the
SINV-derived 5' and 3' UTRs, the subgenomic promoter and nsPs, but
the structural genes were VEEV TRD-specific and had additional
mutations, adapting this virus to efficient growth in BHK-21
cells.
RNA Transcriptions
[0091] Plasmids were purified by centrifugation in CsCl gradients,
and linearized using the MluI restriction site located downstream
of the poly(A) sequence of the VEEV replicons and helper-encoding
plasmids. RNAs were synthesized by using SP6 RNA polymerase in the
presence of a Cap analog. The yield and integrity of transcripts
were analyzed by gel electrophoresis under non-denaturing
conditions. RNA concentration was measured on a Fluor Chem imager
(Alpha Innotech), and transcription reactions were used for
electroporation without additional purification.
Packaging of the Replicons
[0092] BHK-21 cells were co-electroporated with the
in-vitro-synthesized SINV replicon and BBdelSL2/C and BBdel/SL2/GI
helper RNAs or by VEEV replicon and Hvee/C and Hvee/GI helper RNAs
(56). The indicated helper RNA encode either capsid or viral
glycoproteins and contain no packaging signal(s). They are packaged
into viral particles very inefficiently. Packaged replicons were
harvested at 24-30 h post electroporation. Titers of the
replicon-containing, infectious virus particles were determined by
infecting BHK-21 cells with serially diluted samples. Cells were
further incubated for 16 h at 30.degree. C. in a CO.sub.2
incubator, and numbers of infected cells were determined under an
inverted, fluorescent microscope by counting 4xTomato-, Cherry- or
GFP-positive cells. Titers of the infectious VEEV TC-83 and
VEEV/nsP2GFP were determined using the standard plaque assay.
Analysis of Protein Synthesis
[0093] BHK-21 cells were seeded into six-well Costar plates at a
concentration of 5.times.10.sup.5 cells/well. After 4 h incubation
at 37.degree. C. in 5% CO.sub.2, they were infected at an MOI of 20
PFU/cell or 20 infectious units (inf.u)/cell of viruses or packaged
replicons, respectively, in 500 ml of alpha MEM supplemented with
1% FBS at room temperature for 1 h with continuous shaking. The
medium was then replaced by a corresponding complete medium, and
incubation continued at 37.degree. C. At 8 h post infection, the
cells were washed three times with phosphate-buffered saline (PBS)
and then incubated for 30 minutes at 37.degree. C. in 0.8 ml of
DMEM medium lacking methionine, supplemented with 0.1% FBS and 20
mCi/ml of [.sup.35S]methionine. After this incubation, cells were
scraped into the media, pelleted by centrifugation and dissolved in
200 ml of standard protein loading buffer and loaded onto sodium
dodecyl sulfate-10% polyacrylamide gels. After electrophoresis,
gels were dried and autoradiographed.
Microscopy
[0094] BHK-21 cells were seeded on glass chamber slides (Nunc) and
infected or co-infected by the packaged replicons for 1 h.
Incubation was continued for 8 h at 37.degree. C. in a CO.sub.2
incubator. Then cells were fixed in phosphate buffered saline
(PBS), supplemented with 4% formaldehyde, and the distribution of
the proteins was analyzed on a Zeiss LSM510 META confocal
microscope using a 63.times.1.4NA oil immersion planapochromal
lens. For staining of VEEV nsP2, cells were permeabelized with 0.5%
Triton X-100 and stained with anti-VEEV nsP2 antibodies, provided
by AlphaVax (Research Triangle Park, N.C.) (used at 1:2000
dilution). After staining with AlexaFluor 488-labeled secondary
antibodies (Invitrogen), cells were analyzed via confocal
microscopy. These goat anti-VEEV nsP2 antibodies demonstrated
considerable staining of the nuclei in the mock-infected cells.
Therefore, before immunostaining, they were pre-absorbed on the
lysate of BHK-21 cells. 2.times.10.sup.7 cells were used for
pre-adsorption of 1 ml of the antiserum. Cells were pelleted by
low-speed centrifugation, permeabelized with 0.5% Triton-X100 and
additionally homogenized through a syringe needle. Pre-absorption
was performed for 1 h at room temperature with continuous shaking.
Then cell debris was removed by centrifugation at 16,000.times.g
and the supernatant was used for cell staining as described above.
Staining with affinity-purified, rabbit anti-SINV nsP2 (1:2000) and
anti-HA (1:200) (Covance) antibodies was performed using the same
protocol, but the pre-adsorption step was omitted.
Example 16
Results
[0095] VEEV infection inhibits nuclear import. The presence of
C.sub.VEE on the nuclear membrane was identified suggesting the
possibility that this protein might affect nucleocytoplasmic
trafficking, and, thus, interfere with the expression of cellular
genes. Therefore, an experimental system for analysis of inhibition
of the nuclear import was developed by designing a VEEV replicon
encoding a high-molecular-weight fluorescent protein that could not
translocate into the nucleus by passive diffusion and whose
presence in the nucleus could be achieved only by active,
importin-dependent transport through the nuclear pores. It was
believed that only the proteins having a molecular weight below
40-50 kDa could migrate into the nuclei by passive diffusion, which
is mediated by different channels in the nuclear pore complex
(NPC). However, the maximum size of the proteins that are capable
of diffusing through the NPC could be larger than 60 kDa.
Therefore, a 4xTomato protein which had a molecular weight of 109.5
kDa was designed, and it was cloned into the VEEV replicon under
control of the subgenomic promoter. Upon delivery into BHK-21 cells
by electroporation, the expressed 4xTomato was detected only in the
cytoplasm (FIG. 10A (1A of paper). Then, in the following
experiments, this protein was fused with a variety of the NLS
sequences. These modified genes were also cloned into the VEEV
replicon (VEErep) under control of the subgenomic promoter, and the
replicons were packaged into infectious viral particles by using
the previously described two-helper system.
[0096] The first tested expression cassette contained a triple SV40
TAg-derived NLS (VEErep/4xTomato-3xNLS) (FIG. 10B (FIG. 1B of
paper)), which is known to interact with importin-a/b complexes,
mediating nuclear import of about 57% of the cellular proteins. In
initial experiments, that inhibition of nuclear import is a
biological phenomenon that occurs in VEEV-infected cells was
demonstrated. Therefore, BHK-21 cells were either infected with
packaged VEErep/4xTomato-3xNLS replicon (FIG. 10B) or co-infected
with this replicon and VEEV TC-83 (FIG. 10C). In the cells infected
only with packaged replicon, the 4xTomato-3xNLS protein accumulated
to high concentrations in the cell nuclei (FIG. 10B), and in some
cells, formed additional aggregates in the cytoplasm. Co-infection
with VEEV TC-83 completely blocked the translocation of the protein
to the nucleus (FIG. 10C). At 8 h post infection, the
4xTomato-3xNLS was detected only in the cytoplasm of the infected
cells. These experiments demonstrated that VEEV infection inhibits
the nuclear import, mediated by SV40 TAg-derived NLS. The ability
of the 4xTomato-3xNLS to accumulate in the nucleus, when expressed
from VEEV replicon, also indicated that VEEV nsPs do not block
nuclear import.
[0097] VEEV capsid inhibits multiple nucleocytoplasmic transport
pathways. The above-described experiments indicated that VEEV
structural proteins function in the inhibition of nuclear import.
Moreover, it was reasonable to expect that the VEEV capsid, but not
the envelope glycoproteins, plays a critical role in this process.
Therefore, in successive experiments, whether C.sub.VEE affects
nucleocytoplasmic trafficking was examined, and it was
distinguished whether it inhibits only the receptor-mediated
nuclear import pathways or blocks passive diffusion of small
proteins as well. Inhibition of active, so-called classical nuclear
import pathway was analyzed using the above-described VEEV
replicon, expressing the 4xTomato-3xNLS reporter protein (FIG.
11A). The second replicon encoded a small GFP protein (having a
molecular weight of 27 kDa) under the control of the subgenomic
promoter, and another subgenomic promoter was driving the
expression of the VEEV capsid. GFP was used for two reasons: i) its
expression in the cells indicated the presence of the
C.sub.VEE-expressing replicon, and ii) the analysis of the
intracellular distribution of the GFP provided information about
whether passive diffusion of small proteins to the nucleus was
affected by the co-expressed, tested proteins. The rationale for
application of VEEV replicons for expressing capsid and its
derivatives instead of using more traditional RNA polymerase II
promoter-based expression cassettes was based on the fact that that
VEEV- and EEEV-derived capsids efficiently inhibit nuclear
transcription. Therefore, their expression from plasmid DNA-based
cassettes could be problematic.
[0098] Both C.sub.VEE- and 4xTomato-3xNLS-coding replicons were
packaged into infectious viral particles, and BHK-21 cells were
infected separately with VEErep/4xTomato-3xNLS and
VEErep/C.sub.VEE/GFP or with both replicons together. In the
VEErep/C.sub.VEE/GFP-infected cells (see FIGS. 11A-D), GFP was
distributed in the cytoplasm and the nucleus, indicating that
C.sub.VEE expression did not noticeably affect the passive
diffusion of GFP, and, most likely, other small proteins through
the nuclear pores. As described above, the 4xTomato-3xNLS was
concentrated in the nuclei of the cells, infected with the
VEErep/4xTomato-3xNLS-expressing replicons. However, when the same
protein was produced in the cells, co-infected with
C.sub.VEE-encoding replicons, it was no longer transported into the
nucleus and remained only in the cytoplasm. This was a strong
indication that C.sub.VEE inhibits at least an
importin-a/b-dependent nuclear import pathway, which is considered
to be responsible for translocation into the nucleus of more than
50% of the proteins. IC.sub.VEE expression from
VEErep/C.sub.VEE/GFP and the expression of reporter protein
4xTomato-3xNLS and C.sub.VEE derivatives, fused with GFP, was
additionally confirmed by metabolic labeling of the cell proteins
with [.sup.35S]methionine, followed by analysis of the cell
extracts by gel electrophoresis (FIG. 12). The proteins of interest
were readily detectable on gels, and expressed at levels comparable
to those of capsids, expressed by replicating viruses. To rule out
a possibility that inhibition of nuclear import is a synergistic
effect of VEEV nsP(s) and capsid functions, 4xTomato-3xNLS and VEEV
capsid were also expressed from the T7 promoter/EMCV IRES-dependent
cassettes in i) BHK-21 cells-derived cell line expressing T7
DNA-dependent RNA polymerase and ii) BHK-21 cells infected with a
vaccinia virus recombinant expressing the same enzyme. In both
expression systems, VEEV capsid efficiently inhibited nuclear
import of 4xTomato-3xNLS. Thus, the capsid protein itself can
interfere with nuclear import.
[0099] However, the detected blockage of importin-a/b-dependent
nuclear import does not necessarily indicate that the transport of
all of the proteins is affected. Therefore, the function of two
other, M9- and histone H2b-specific, pathways were evaluated in the
presence of C.sub.VEE. The M9 NLS, derived from hnRNPA1 is
recognized by transportin that mediates nuclear transport of the
RNA-binding proteins. H1stone H2b utilizes multiple nuclear import
pathways, mediated by importin-a/b complex, transportin, importin
5, importin 7, importin 7/importin b complex and importin 9.
Application of this reporter allows detection of alterations in the
nuclear import pathways other than SV40 TAg NLS- or
M9-dependent.
[0100] Thus, both M9- and the entire mouse H2b histone-coding
sequences were cloned into the carboxy terminus of the 4xTomato
gene. Modified replicons, [0101] VEErep/4xTomato-M9 and
VEErep/4xTomato-H2b were packaged into the infectious viral
particles, and, after infection of BHK-21 cells, both peptides
demonstrated efficient functioning in the translocation of the
4xTomato into the nucleus (FIG. 11D). However, the M9 NLS was
noticeably less efficient than was the triple SV40 TAg NLS and H2b
protein, and a significant fraction of the 4xTomato-M9 reporter
remained in the cytoplasm (FIG. 11D). In the cells, co-infected
with the 4xTomato-encoding replicons and the replicon expressing
C.sub.VEE, both 4xTomato-M9 and 4xTomato-H2b proteins were not
transported into the nucleus (FIGS. 11B and C). These data indicate
that C.sub.VEE is capable of inhibiting nuclear import mediated by
at least three different types of NLSs, and, most likely, its
presence in the cells strongly affects the entire nuclear
import.
[0102] SINV capsid expression does not affect nuclear import. The
capsid protein of the Old World alphaviruses, SINV and SFV, is
incapable of inhibiting cellular transcription and causing CPE, in
spite of being present in the nuclei. To further dissect the
differences between the New World and the Old World alphavirus
capsid functions in virus-host cell interactions, the SINV-specific
capsid (C.sub.SIN) gene was cloned into the VEEV replicon and
co-infected BHK-21 cells with this packaged VEErep/C.sub.SIN/GFP
and the described above VEErep/4xTomato-3xNLS replicons. In
contrast to C.sub.VEE, the SINV-specific capsid had no noticeable
effect on the translocation of 4xTomato-3xNLS into the nuclei (FIG.
13A). The latter protein was transported to the nucleus as
efficiently as in the cells infected with VEErep/4xTomato-3xNLS
replicon alone. Thus, the Old World alphavirus SINV-specific capsid
either causes only a minor effect on the nuclear import,
(undetectable herein), or does not have this activity. Thus, its
inability to interfere with the nuclear import correlates with the
lack of C.sub.SIN-specific transcriptional shutoff.
[0103] The VEEV capsid-specific N-terminal peptide plays a critical
role in blocking the nucleocytoplasmic transport. The present
invention identified an amino terminal peptide in C.sub.VEE that
plays a critical role in CPE development and induction of
transcriptional shutoff. This peptide, located between aa 32 and 69
of C.sub.VEE, has two short, distinct domains: the aa 33-51, which
were previously shown to fold into an a-helix secondary structure,
and the downstream, positively charged aa sequence that likely
contains a functional NLS. The C.sub.VEE32-68 peptide, fused with
GFP, accumulates on nuclear membrane and causes CPE as efficiently
as the entire C.sub.VEE. These data suggested that this fragment
might play a critical role in the C.sub.VEE-mediated inhibition of
the nucleocytoplasmic traffic.
[0104] To demonstrate this, the amino terminal fragment of
C.sub.VEE (aa 1-68), fused with GFP (VEErep/C.sub.VEE1-68-GFP) was
cloned into the VEEV replicon (FIG. 13B). The aa 1-68 sequence was
applied instead of a minimal peptide to promote similar expression
of this particular fusion protein and other constructs. The second
replicon encoded C.sub.VEE-GFP fusion, in which a short sequence in
the C.sub.VEE (aa 58-85) was changed by two frame-shift mutations
(FIG. 13C). This C.sub.VEEfrsh-GFP fusion, was found to be
noncytopathic and incapable of inhibiting cellular transcription.
VEEV replicon, encoding the entire wt C.sub.VEE, fused with GFP
(FIG. 4D), was used as a positive control.
[0105] All of the replicons were packaged into infectious viral
particles and used for co-infection of BHK-21 cells, together with
the packaged VEErep/4xTomato-3xNLS. Expression of C.sub.VEE1-68-GFP
inhibited translocation of 4xTomato-3xNLS to the nucleus (FIG.
13B), as well as did the expression of the control C.sub.VEE-GFP
fusion (FIG. 13D). (C.sub.VEE1-68-GFP reproducibly formed small
aggregates that might reflect its .about.2-fold higher level of
expression, compared to those of other chimeric proteins). However,
in the cells, co-infected with C.sub.VEEfrsh-GFP-producing replicon
and VEErep/4xTomato-3xNLS, transport of the 4xTomato-3xNLS to the
nucleus was not altered (FIG. 13C), despite the expression of both
C.sub.VEE-GFP and C.sub.VEEfrsh-GFP at very similar rates and to
similar levels, which were very similar to those shown for other
proteins in FIG. 12. Thus, the amino terminal sequence of C.sub.VEE
inhibits the classical nuclear import pathway. The previously
described, frame-shift mutation in C.sub.VEE peptide that makes
this protein incapable of translocation into the nucleus and
affects its ability of causing CPE and inhibiting cellular
transcription also makes C.sub.VEE unable to block nuclear
import.
[0106] VEEV nsP2 accumulates mainly in the cytoplasm. One of the
nonstructural proteins, nsP2, of the Old World alphaviruses (SINV
and SFV) is known to be present at a high concentration in the cell
nuclei, where it functions in transcription inhibition. In the case
of SINV and, most likely, of SFV, this translocation is indirectly
supported by the inability of the capsid protein to interfere with
the nuclear import. However, the newly described function of
C.sub.VEE suggests that translocation of VEEV nsP2 into the nucleus
might be problematic, and, in contrast to its SINV-specific
counterpart, this protein is likely to accumulate in the cell
nuclei at a low concentration. Data indirectly supported the
possibility that VEEV nsP2 does not play a critical role in
modification of the nuclear function: i) the VEEV (and EEEV)
replicons were incapable of downregulating cellular transcription
to a level incompatible with cell survival, and persistently
replicated in some of the cell lines of vertebrate origin; ii) the
VEEV variants having either the capsid or entire subgenomic RNA
replaced by SINV-specific counterparts were dramatically less
cytopathic than were the parental VEEV and could not interfere with
the cellular antiviral response. These chimeric viruses either
persisted in the cells, defective in the IFN-a/b response, or were
cleared from the cells having functional IFN-a/b signaling.
[0107] To further understand the differences between the New World
and the Old World alphavirus nsP2 activities in modification of the
nuclear function, the distribution of VEEV nsP2, produced by
different expression cassettes was analyzed and how C.sub.VEE
interferes with the nuclear import of VEEV (and SINV) nsP2 was
evaluated.
[0108] Recent data suggests that VEEV nsP2 and its fragments can be
detected in the nuclei. Therefore, in order to generate definitive
results, multiple cell lines and a variety of expression constructs
were used to analyze the intracellular distribution of this
protein. Initially, 3 cell lines, BHK-21, HEK293 and NIH 3T3 cells,
were infected with VEEV TC-83 virus. The available VEEV
nsP2-specific antibodies demonstrated significant staining of the
nuclei in the mock-infected cells, and were pre-adsorbed in the
lysate of the BHK-21 cells. After the pre-adsorption, staining of
the mock-infected cells was not detectable (FIG. 14D).
[0109] By 8 h post infection with VEEV TC-83, nsP2 was present in
the nuclei of all of the tested cell lines at a very low
concentration (FIGS. 14A, B and C). At later times, this protein
was found at a higher concentration in the nuclei of a small
percentage of the infected cells, which were already on very
advanced stages of apoptosis. Thus, the presence of C.sub.VEE, the
VEEV nsP2 is unlikely to play a major role in cellular nuclei,
because of its low presence in this compartment.
[0110] The effect of C.sub.VEE expression on the
compartmentalization of nsP2 of different origins was evaluated.
BHK-21 cells were infected either i) with the wt SINV, which
encodes capsid protein that does not affect nuclear import (FIG.
15A), or ii) with SIN/VEEV chimeric virus that expresses all of the
VEEV structural proteins (FIG. 15B). In the SINV-infected cells,
SINV nsP2 was distributed both in the cytoplasm and nuclei (FIG.
15A), and in the SIN/VEEV-infected cells, nsP2 was detected only in
the cytoplasm (its concentration in this compartment was noticeably
higher than that in SINV-infected cells) (FIG. 15B). This indicated
that C.sub.VEE expression likely interferes with SINV nsP2
translocation to the nuclear compartment.
[0111] An infectious VEEV TC-83 variant that contained a GFP
insertion in the very amino terminus of nsP2, after aa 3 (FIG. 16)
was designed. In spite of normal processing of the nonstructural
polyprotein and productive virus replication, the nsP2/GFP,
expressed by VEEV/nsP2GFP, was detected only in the cytoplasm of
the infected cells.
[0112] However, the above experiments could not provide a
definitive answer to the question of whether VEEV nsP2 itself had
not developed an ability for translocation into the nucleus, or
whether its cytoplasmic distribution was a result of C.sub.VEE
function in blocking nuclear import. To distinguish between these
possibilities, BHK-21 cells were infected with VEEV replicon,
VEErep/Cherry, that did not encode any structural proteins and
stained the cells with VEEV nsP2-specific antibodies. Almost all of
the nsP2 was detected in the cytoplasm of the replicon-infected
cells (FIG. 17A). In another approach, BHK-21 cells were infected
with packaged SINV replicon that had a mutation in the nsP2/nsP3
cleavage cite and encoded the HA-tagged VEEV nsP2 under the control
of the subgenomic promoter (FIG. 8B). This cleavage cite mutant was
incapable of P23 processing, and, thus, the unprocessed SINV nsP2
remained in the cytoplasm and could not interfere with VEEV nsP2
transport into the nucleus. In the cells, infected with
SINrep2V/nsP2VEE-HA, the HA-tagged nsP2 was also detected only in
the cytoplasm (FIG. 17B). Taken together, the results of the
experiments strongly suggested that, i) compared to SINV or SFV
nsP2, the VEEV-specific counterpart had a dramatically reduced
ability for translocation into the nucleus, and ii) its presence in
the nuclei at very low concentrations was not only the effect of
C.sub.VEE functioning in modification of the nucleocytoplasmic
traffic, but also a result of the inability of VEEV nsP2 itself for
import into the nucleus.
[0113] C.sub.VEE does not affect nuclear import in mosquito cells.
The characteristic feature of alphavirus infection in the mosquito
cells is the development of persistent, noncytopathic replication
that mirrors the persistent infection in the mosquito vectors. The
antiviral response in arthropod cells is different from that in the
vertebrate cells, and appears to be not as robust as that in cells
of vertebrate origin. Moreover, inhibition of both nuclear import
and cellular transcription, likely resulting in development of CPE,
could be disadvantageous for persistent virus replication and would
have a negative effect on the viability of infected mosquitoes and
ultimately virus transmission in nature. Thus, C.sub.VEE expression
might have no effect on the nuclear import in mosquito cells.
[0114] Replicons, packaged into a VEEV TC-83-specific envelope,
infected C.sub.7-10 cells inefficiently, and, therefore,
VEErep/4xTomato-3xNLS, VEErep/C.sub.VEE-GFP and
VEErep/C.sub.VEE/GFP replicon genomes were delivered into these
cells by electroporation. Evaluation of the 4xTomato-3xNLS
distribution in the C.sub.VEE-GFP- (FIG. 18A) and C.sub.VEE-
expressing cells (FIG. 18B) strongly indicated that C.sub.VEE had
no noticeable effect on the translocation of the 4xTomato-3xNLS
protein into the cell nuclei. Regardless of the presence of
C.sub.VEE and C.sub.VEE-GFP, the 4xTomato-3xNLS was efficiently
transported into the nucleus. Thus, this failure to inhibit nuclear
import correlates with the inability of the virus to cause CPE and
appears to be one of the important determinants of persistent
replication in mosquito cells and mosquito vectors.
Discussion
[0115] Replication of alphavirus-specific RNAs, translation of the
viral proteins, and release of infectious particles do not require
a nuclear function. Viral replication can proceed for a long time
in the presence of ActD or even in the anucleated cells.
Nevertheless, interaction of replicating virus with host cell
nuclei plays a critical role in the infectious process and strongly
determines pathogenesis on the molecular, cellular and organismal
levels. As do other viruses, alphaviruses appear to induce an
innate intracellular antiviral response (virus-induced cell
response) that is mediated by cellular pathogen recognition
receptors (PRRs). The latter response leads to the induction of
cell signaling, initiating an antiviral state in the
as-yet-uninfected cells and activation of an antiviral reaction in
the already infected cells. These processes interfere with
productive viral replication and dissemination of the infection.
Alphaviruses, in turn, developed mechanisms aimed at strong
modification of cell biology and/or development of robust, rapid
CPE. In these processes, inhibition of cellular translational and
transcriptional machineries appears to play a critical role. These
modifications of the intracellular environment are likely to be
multicomponent phenomena and determined by different viral
nonstructural and structural proteins. The accumulated data
strongly indicate that both the Old and New World alphaviruses are
capable of downregulating cellular transcription, but utilize very
different mechanisms for achieving this goal. The Old World
alphavirus-specific nonstructural protein nsP2 is a key player in
the inhibition of both cellular messenger and ribosomal RNAs
synthesis. This protein causes CPE development, either when being
expressed in the context of ns polyprotein, or alone from different
expression cassettes. On the other hand, the New World alphaviruses
VEEV and EEEV use capsid protein, but not the nsP2, for causing the
same phenomenon. This structural protein is distributed both in the
cytoplasm and nuclei of the infected cells, and downregulates RNA
polymerase I- and II-dependent transcription, and its expression
ultimately leads to CPE development. This function of VEEV capsid
is determined by a short amino terminal peptide capable of
inhibiting transcription even when being fused to other proteins.
Modifications of the active peptide in the VEEV genome made virus
dramatically less cytopathic and attenuated in vivo without having
a strong effect on its replication rates (or no effect at all for
some of the constructs).
[0116] The distinguishing features of the VEEV capsid-dependent
transcriptional shutoff include its slow rates of development and a
lack of dependency on the capsid-associated protease activity.
These data strongly indicated stoichiometric rather than enzymatic
modes of capsid functioning. Moreover, a significant fraction of
C.sub.VEE was found to be associated with the nuclear membrane and
its distribution was reminiscent of that of the nuclear pore
complexes. The C.sub.VEE32-68 peptide fused with GFP demonstrated a
similar distribution and was also capable of blocking cellular
transcription. Taken together, the results indicated that C.sub.VEE
(and its active C32-68 peptide) might be involved not only in
downregulation of cellular transcription, but also in modification
of the NPC and inhibition of nucleocytoplasmic traffic.
[0117] The present invention showed the function of several nuclear
import pathways, mediated by different importins, and found that
all of them were inhibited by the C.sub.VEE within 8 h post
infection. (Notably, for expression of this protein, the most
adequate system, VEEV replicons, was used which express capsid in
appropriate cellular compartments and at natural concentrations.)
The C.sub.VEE1-68 peptide, fused with GFP, demonstrated a very
similar activity, and C.sub.VEEfrsh-GFP, which does not inhibit
cellular transcription and is undetectable in the cell nuclei and
on the nuclear membrane, had no effect on the nucleocytoplasmic
traffic. Thus, accumulation of C.sub.VEE in the nuclear envelope
strongly correlated with the inhibition of nuclear import. In
infected cells, nucleocytoplasmic traffic is strongly modified, and
a great fraction of the newly synthesized cellular proteins is not
transported into the nucleus. Among the RNA viruses, blockage of
nuclear import is not a new phenomenon. Inhibition of nuclear
transport has been described for VSV, poliovirus, rhinovirus and
cardiovirus. The VSV matrix protein interacts with the nucleoporin
Nup98 and export receptor Rae 1. Thus, M protein accumulates in the
NPCs, in which it efficiently inhibits Rae 1-mediated mRNA nuclear
export and slows the rate of the nuclear import through the
importin a/b1-dependent pathway. Interestingly, VSV also
efficiently inhibits cellular transcription, but the correlation
between inhibition of nucleocytoplasmic traffic and downregulation
of transcription has not been investigated. Two members of the
Picornoviridae family, poliovirus and rhinovirus, employ different
mechanisms and inhibit nuclear import by degrading the nucleoporins
Nup62 and Nup153 by virus-encoded proteases. The same proteases
also process the transcription factors. VEEV and, most likely,
other New World alphaviruses employ a mechanism that appears to be
based on interaction of the capsid protein with NPC. Moreover,
functioning of C.sub.VEE1-68-GFP fusion in inhibition of nuclear
import suggests that capsid might function without proteolytic
cleavage of the Nups.
[0118] The Old World alphaviruses, SINV and SFV, produce capsid
proteins that neither block nuclear import, nor interfere with
cellular transcription, and do not induce CPE. Consequently, one of
the characteristic features of such infection is the accumulation
of large amounts of nsP2 in the cell nuclei. The newly described
function of C.sub.VEE in the inhibition of the nucleocytoplasmic
transport suggested that VEEV nsP2 had to be present in the cell
nuclei at lower concentration, and unlikely to play an important
role in this compartment. Moreover, VEEV nsP2 itself was found to
be inefficient in its translocation to the nucleus. It was detected
almost exclusively in the cytoplasm not only in the virus-infected
cells, but also when produced by VEEV replicon, and expressed from
other vectors as GFP-nsP2 or nsP2-HA tag fusions. Such VEEV nsP2
compartmentalization indirectly supports the finding that VEEV
replicons, expressing no capsid, caused a less efficient CPE than
did similar SINV- and SFV-based constructs and were capable of
establishing a persistent replication in the mammalian cells. This
was an indication that VEEV- and EEEV-specific nsP2 and other nsPs
did not cause as strong, negative effect on cellular biology as did
the nsPs of the Old World alphaviruses. Notably, the putative
nsP2-specific NLS (that was described in the SFV nsP2) is replaced
in VEEV nsP2 by a different aa sequence. This peptide might still
function as an NLS, and the mutations in this sequence affect virus
and replicon RNA replication. However, as shown for SFV, mutations
in this particular peptide could strongly affect the rates of the
ns polyprotein processing and/or other nsP2 functions in the RNA
replication. Therefore the existence of monopartite NLS in VEEV
nsP2 remains questionable. Nevertheless, it is possible that this
nonstructural protein, produced by replicating VEEV, has some
function in modification of the nucleocytoplasmic traffic because
of its recently described interaction with karyopherin 1.
[0119] Interestingly, C.sub.VEE was found to be incapable of
blocking nuclear import in mosquito cells. Large, NLS-containing
protein 4xTomato-3xNLS was transported into the cell nuclei
regardless of the presence of C.sub.VEE in the same cells. Thus,
the inability of VEEV capsid to interfere with nucleocytoplasmic
trafficking provides a plausible explanation for the noncytopathic
phenotype of the VEEV in the mosquito cells. However, this is not
the only critical difference between virus replication in the cells
of the vertebrate and invertebrate origin.
[0120] In conclusion, the present invention demonstrated that i)
C.sub.VEE efficiently inhibits nuclear import, but does not affect
passive diffusion of small proteins; ii) the amino terminal
sequence Of C.sub.VEE interferes with nuclear import as efficiently
as does the entire C.sub.VEE; iii) the capsid protein of the Old
World alphavirus, SINV, or C.sub.VEE with previously defined
frame-shift mutations (C.sub.VEEfrsh), which makes it incapable of
transcription inhibition, have no detectable effect on
nucleocytoplasmic trafficking; iv) Inhibition of the NPC function
is one of the critical mechanisms, which the New World alphaviruses
employ for the downregulation of cellular transcription and CPE
development. v) C.sub.VEE does not noticeably interfere with
NPC-mediated nuclear import in the mosquito cells, and this might
play a critical role in the ability of the virus to develop a
persistent, life-long infection in mosquito vectors.
[0121] The following references were cited herein: [0122] 1.
Aguilar, P. V. et al. 2007, J Virol 81:3866-76. [0123] 2.
Alevizatos, A. C. et al. 1967, Am J Trop Med Hyg 16:762-8. [0124]
3. Berge, T. O. et al. 1961, Am. J. Hyg 73:209-218. [0125] 4.
Black, B. L. et al 1993, J Virol 67:4814-21. [0126] 5. Bredenbeek,
P. J. et al. 1993, J. Virol. 67:6439-6446. [0127] 6. Burke, D. S.
et al. 1977, J Infect Dis 136:354-359. [0128] 7. Choi, H. K. et al.
1991, Nature 354:37-43. [0129] 8. Faria, P. A. et al. 2005, Mol
Cell 17:93-102. [0130] 9. Frolova, E. I. et al. 2002, J Virol
76:11254-11264. [0131] 10. Garcia-Tamayo, J. et al. 1979, J Pathol
128:87-91. [0132] 11. Garmashova, N. et al. 2006, J Virol
80:5686-96. [0133] 12. Garmashova, N., et al. 2007, J Virol
81:2472-84. [0134] 13. Gorchakov, R. et al. 2005, J Virol
79:9397-409. [0135] 14. Griffin, D. 1986. Alphavirus Pathogenesis
and Immunity, p. 209-250. In M. Schlesinger (ed.), The Togaviridae
and Flaviviridae. Plenum Press, New York. [0136] 15. Griffin, D. E.
2001. Alphaviruses, p. 917-962. In D. Knipe and P. Howley (ed.),
Fields' Virology, Fourth Edition. Lippincott, Williams and Wilkins,
New York. [0137] 16. Gustin, K. E. 2003 Virus Res 95:35-44. [0138]
17. Gustin, K. E., and P. Sarnow. 2001, Embo J 20:240-9. [0139] 18.
Gustin, K. E., and P. Sarnow. 2002, J Virol 76:8787-96. [0140] 19.
Hahn, C. S. et al. 1985, Proc. Natl. Acad. Sci. USA 82:4648-4652.
[0141] 20. Hahn, C. S., and J. H. Strauss. 1990, J. Virol.
64:3069-3073. [0142] 21. Henderson, B. E. et al. 1971, Am. J.
Epidemiol. 93:194-205. [0143] 22. Imreh, G., and E. Hallberg. 2000,
Exp Cell Res 259:180-90. [0144] 23. Johnson and Martin. 1974, Adv.
Vet. Sci. Comp. Med. 18:79-116. [0145] 24. Kinney, R. M. et al.
1993, J. Virol. 67:1269-1277. [0146] 25. Kinney, R. M. et al. 1989,
Virology 170:19-30. [0147] 26. Lemm, J. A. et al. 1990, J. Virol.
64:3001-3011. [0148] 27. Leon, C. A. 1975, Int J Epidemiol
4:131-40. [0149] 28. Lidsky, P. V. et al. 2006, J Virol 80:2705-17.
[0150] 29. Liljestrom, P. et al. 1991, J. Virol. 65:4107-4113.
[0151] 30. Naim, B. et al. 2007, J Biol Chem 282:3881-8. [0152] 31.
Perera, R. et al. 2003, J Virol 77:8345-53. [0153] 32. Perera, R.
et al. 2001, J Virol 75:1-10. [0154] 33. Petersen, J. M. et al.
2001, Proc Natl Acad Sci USA 98:8590-5. [0155] 34. Petersen, J. M.
et al. 2000, Mol Cell Biol 20:8590-601. [0156] 35. Petrakova, O. et
al. 2005, J Virol 79:7597-608. [0157] 36. Pittman, P. R. et al.
1996, Vaccine 14:337-43. [0158] 37. Porter, F. W. et al. 2006, Proc
Natl Acad Sci USA 103:12417-22. [0159] 38. Rice, C. M. et al. 1987,
J. Virol. 61:3809-3819. [0160] 39. Rice, C. M., and J Strauss 1981,
Proc. Natl. Acad. Sci. USA 78:2062-2066. [0161] 40. Rico-Hesse, R.
et al 1995, Proc. Natl. Acad. Sci. USA 92:5278-5281. [0162] 41.
Rivas, F. et al. 1997, J Infect Dis 175:828-32. [0163] 42. Strauss,
J. H., and E. G. Strauss. 1994, Microbiol. Rev. 58:491-562. [0164]
43. Takkinen, K. 1986, Nucleic Acids Res. 14:5667-5682. [0165] 44.
Volchkov, V. E. et al. 1991, Mol. Genet. Mikrobiol. Virusol.
5:8-15. [0166] 45. Volkova, E. et al. 2006, Virology 344:315-27.
[0167] 46. von Kobbe, C. et al. 2000, Mol Cell 6:1243-52. [0168]
47. Weaver, S. C., and A. D. Barrett. 2004, Nat Rev Microbiol
2:789-801. [0169] 48. Weaver, S. C. et al. 1994, J Virol 68:158-69.
[0170] 49. Weaver, S. C. et al. 1996, Lancet 348:436-40. [0171] 50.
Wengler, G. et al. 1992, Virology 191:880-8.
[0172] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. These patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually incorporated by
reference.
[0173] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. It will be apparent to those skilled in the art that
various modifications and variations can be made in practicing the
present invention without departing from the spirit or scope of the
invention. Changes therein and other uses will occur to those
skilled in the art which are encompassed within the spirit of the
invention as defined by the scope of the claims.
Sequence CWU 1
1
5139PRTAlphaviruscapsid fragment sequence of Venezuelan equine
encephalitis virus with deletion after Arg 24 1Thr Asp Pro Phe Leu
Ala Met Gln Val Gln Glu Leu Thr Arg Ser1 5 10 15Met Ala Asn Leu Thr
Phe Lys Gln Arg Arg Asp Ala Pro Pro Glu 20 25 30Gly Pro Ser Ala Lys
Lys Pro Lys Lys 35238PRTAlphaviruscapsid fragment sequence of
eastern equine encephalitis virus with deletion after Arg 24 and
Arg 36 2Phe Arg Pro Pro Leu Ala Ala Gln Ile Glu Asp Leu Arg Arg
Ser1 5 10 15Ile Ala Asn Leu Thr Leu Lys Gln Arg Ala Pro Asn Pro Pro
Ala 20 25 30Gly Pro Pro Ala Lys Arg Lys Lys
35340PRTAlphaviruscapsid fragment sequence of Sindbis virus 3Ala
Arg Asn Gly Leu Ala Ser Gln Ile Gln Gln Leu Thr Thr Ala1 5 10 15Val
Ser Ala Leu Val Ile Gly Gln Ala Thr Arg Pro Gln Pro Pro 20 25 30Arg
Pro Arg Pro Pro Pro Arg Gln Lys Lys 35 40439PRTAlphaviruscapsid
fragment sequence of Semliki Forest virus with deletion after Asn
24 4Val Pro Asp Phe Gln Ala Gln Gln Met Gln Gln Leu Ile Ser Ala1 5
10 15Val Asn Ala Leu Thr Met Arg Gln Asn Ala Ile Ala Pro Ala Arg 20
25 30Pro Pro Lys Pro Lys Lys Lys Lys Thr 35512PRTartificial
sequencelinker sequence for a fusion protein 5Gly His Gly Thr Gly
Ser Gly Gly Ser Gly Ser Ser1 5 10
* * * * *