U.S. patent application number 10/150283 was filed with the patent office on 2003-11-27 for rna silencing in animals as an antiviral defense.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Ding, Shou-Wei, Li, Hong-Wei, Li, Wan-Xiang.
Application Number | 20030219407 10/150283 |
Document ID | / |
Family ID | 29548328 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030219407 |
Kind Code |
A1 |
Ding, Shou-Wei ; et
al. |
November 27, 2003 |
RNA silencing in animals as an antiviral defense
Abstract
The present invention provides recombinant DNA constructs for
inactivation of viral or endogenous genes in a cell, wherein the
construct comprises viral sequence sufficient to activate RNA
silencing. In another aspect, the invention provides methods for
identifying RNA silencing suppressors by sequence analysis and
functional tests. In yet another aspect, the invention provides a
method for identifying inhibitors of RNA silencing suppressors. In
still other aspects, the invention comprises methods for
identifying genes in the antiviral RNA silencing pathway, enhancers
of the antiviral pathway, and methods of treating or preventing
viral infections using enhancers of the pathway.
Inventors: |
Ding, Shou-Wei; (Riverside,
CA) ; Li, Hong-Wei; (Riverside, CA) ; Li,
Wan-Xiang; (Riverside, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Regents of the University of
California
12th Floor 1111 Franklin Street
Oakland
CA
94607-5200
|
Family ID: |
29548328 |
Appl. No.: |
10/150283 |
Filed: |
May 15, 2002 |
Current U.S.
Class: |
424/93.2 ;
435/6.13; 514/44A; 536/23.2 |
Current CPC
Class: |
C12N 15/8218 20130101;
C12N 2760/16122 20130101; C12N 15/85 20130101; C12N 15/1131
20130101; C12N 2770/30022 20130101; C12N 2830/002 20130101; C12N
15/86 20130101; C07K 14/005 20130101; C12N 2310/14 20130101 |
Class at
Publication: |
424/93.2 ;
536/23.2; 514/44; 435/6 |
International
Class: |
A61K 048/00; C12Q
001/68; C07H 021/04 |
Goverment Interests
[0001] This work was made with Government support under Grant No.
2002-35319-11537. awarded by the U.S. Department of Agriculture.
The government has certain rights in this invention.
Claims
What is claimed is:
1. A recombinant DNA construct for inactivation of a gene in a
cell, said construct comprising: (a) polynucleotide sequence of a
virus sufficient to activate RNA silencing and (b) the
polynucleotide sequence of said gene.
2. The recombinant DNA construct of claim 1, wherein said construct
is a viral vector capable of infecting said cell.
3. The recombinant DNA construct of claim 1, wherein said construct
is a recombinant vector.
4. The recombinant DNA construct of claim 1, wherein said virus is
FHV.
5. The recombinant DNA construct of claim 4, wherein said construct
further comprises polynucleotide sequences enabling introduction
into said cell.
6. The recombinant DNA construct of claim 1, wherein said cell is
in an animal.
7. The recombinant DNA construct of claim 1, wherein said gene is a
polynucleotide sequence from a heterologous virus.
8. The recombinant DNA construct of claim 1, wherein said gene is a
polynucleotide sequence from an endogenous gene.
9. The recombinant DNA construct of claim 8, wherein expression or
overexpression of said endogenous gene induces a disease or a
medical condition.
10. The recombinant DNA construct of claim 8, wherein said
endogenous gene is an oncogene.
11. A method for identifying a RNA silencing suppressor, said
method comprising: (a) introducing a polynucleotide sequence
encoding FHV-AB2 into an animal cell; (b) introducing a
polynucleotide sequence encoding a candidate RNA silencing
suppressor into said cell; and (c) testing for a rate or extent of
FHV-AB2 RNA accumulation greater than that for a cell not contacted
with said candidate suppressor.
12. The method of claim 11, wherein said RNA silencing suppressor
is from a virus.
13. The method of claim 11, wherein said RNA silencing suppressor
is an endogenous gene.
14. A method for identifying a viral RNA silencing suppressor, said
method comprising identifying a polynucleotide sequence that is an
overlapping gene.
15. The method of claim 14, wherein said gene overlaps a RNA
polymerase gene.
16. A method for identifying an inhibitor of a RNA silencing
suppressor, said method comprising: (a) infecting an animal cell
with FHV; (b) contacting said cell with a candidate inhibitor of a
RNA silencing suppressor; and (c) testing for a rate or extent of
FHV RNA accumulation less than that for a cell not contacted with
said candidate inhibitor of a RNA silencing suppressor.
17. The method of claim 16, wherein said candidate inhibitor is a
small molecule.
18. The method of claim 16, wherein said testing for accumulation
of FHV RNA comprises a method selected from the group consisting
of: visual assays for expressed reporter molecules and Northern
blots.
19. A method for identifying a gene in the antiviral RNA silencing
pathway of an animal, said method comprising: (a) providing an
animal cell expressing a polynucleotide encoding FHV-AB2; (b)
inhibiting the expression of a candidate gene in the antiviral RNA
silencing pathway; and (c) testing for a rate or extent of FHV-AB2
RNA accumulation greater than that for a cell where the expression
of said candidate gene in the antiviral RNA silencing pathway has
not been inhibited.
20. The method of claim 19, wherein said polynucleotide encoding
FHV-.DELTA.B2 further comprises a reporter molecule.
21. The method of claim 20, wherein said reporter molecule is
GFP.
22. The method of claim 19, wherein the expression of said
candidate gene is inhibited by RNA interference, antisense DNA, or
a ribozyme.
23. A method for identifying an enhancer of the antiviral RNA
silencing pathway in an animal, said method comprising: (a)
providing an animal cell infected with FHV; (b) contacting said
cell with a candidate enhancer of the antiviral RNA silencing
pathway; and (c) testing for a rate or extent of FHV RNA
accumulation which is less than that for a cell which is not
contacted with said candidate enhancer.
24. A method for treating or preventing a viral infection in an
animal, said method comprising enhancing the antiviral RNA
silencing pathway by contacting said animal with a compound that
modulates a gene identified by the method of claim 19.
25. The method of claim 24, wherein said gene is AGO2 or an AGO2
homologue in said animal.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0002] NOT APPLICABLE
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] NOT APPLICABLE
BACKGROUND OF THE INVENTION
[0004] RNA interference (RNAi) is a process where introduction of
dsRNA into a cell causes destruction of RNA in a sequence-specific
manner (see, D. Baulcombe, Curr. Biol., 12:R83 (2002); Hutvagner et
al., Curr. Opin. Genet. Dev., 12:225 (2002)) RNAi has been observed
in plants, Neurospora, flies, protozoans, and mice. Available data
show that double-stranded (ds) RNA serves as the initial trigger of
RNA interference and upon recognition, is processed by the Dicer
RNAse into short fragments of 21 nucleotides (nt) in length. These
short interfering (si)RNAs are then incorporated into a
dsRNA-induced silencing complex (RISC) to guide cycles of specific
RNA degradation.
[0005] Recent work has established that in higher plants targeted
degradation of RNA occurs as a natural antiviral response, rather
than simply being a response to artificially introduced or
artificially induced dsRNA. Work by Dougherty and co-workers shows
that virus infection is able to trigger RNA silencing of a
homologous virus-derived transgene in transgenic tobacco (see,
Lindbo et al., Plant Cell, 5:1749-1759 (1993)). The activation of
silencing is accompanied by recovery of the host from the initially
virulent infection so that the new growth is both symptom and
virus-free and is highly resistant to a secondary challenge by the
same virus. This type of RNA-mediated virus resistance (RMVR),
demonstrated conclusively for a number of dicot plant species with
a variety of viruses (see, Waterhouse et al., Trends Plant Sci.,
4:452-457 (1999)), is also functional in monocot plants (see,
Ingelbrecht et al., Plant Physiol., 119:1187-1188 (1999)). This
phenomenon, termed RNA silencing, has been shown to occur via a
similar mechanism as RNA interference. Many plant RNA viruses have
been found to encode efficient suppressors of RNA silencing. One
such suppressor is the 2b protein encoded by cucumber mosaic
cucumovirus (CMV). The idea that Cmv2b functions as a suppressor of
host defense was first proposed based in the finding that Cmv2b is
essential for the development of CMV disease symptoms in its hosts
(see, Ding et al., EMBO J, 14:5762 (1995)). Previous analyses also
indicated that the Cmv2b gene represents a newly evolved gene as
compared to the other four CMV genes (see, Ding et al., Virology,
198:593-601 (1994)), suggesting that the Cmv2b gene is a viral
adaptation to the RNA silencing antiviral defense in plants.
Available evidence also shows that suppression of RNA silencing
plays central role in the induction of viral disease in plants
(see, Ding et al., Curr. Opin. Biotechnol., 11(2):152-156).
[0006] It is well established that cellular and humoral adaptive
immunity based on peptide recognition are defenses employed against
viruses by animals. However, little is known about other antiviral
defenses in animals. In order to develop effective treatments for
viral infections in mammals, it is necessary to identify and
characterize these additional modes of antiviral defense. This
invention fulfills these and other related needs.
BRIEF SUMMARY OF THE INVENTION
[0007] In one aspect, this invention provides recombinant DNA
constructs for inactivation of a gene in a cell, where the
constructs comprise polynucleotide sequence of a virus sufficient
to activate RNA silencing and the polynucleotide sequence of the
gene to be inactivated. In some embodiments, the sequence which
activates RNA silencing is from the flock house virus (FHV). The
constructs can either be infectious viral vectors or recombinant
vectors. In certain embodiments, the constructs further comprise
polynucleotide sequences enabling introduction into certain types
of cells. In addition to inactivating genes in cells, the
recombinant constructs of this invention can also be used to
inactivate genes in a whole animal.
[0008] The gene inactivated by the recombinant constructs of this
invention can be from a heterologous virus. Alternatively, the
inactivated gene can be an endogenous gene. In certain embodiments,
the endogenous gene is one where expression or overexpression of
the endogenous gene induces a disease or a medical condition. The
inactivated endogenous gene can be an oncogene.
[0009] In another aspect, the invention provides methods for
identifying a RNA silencing suppressor. Typically, the methods
comprise (a) introducing a polynucleotide sequence encoding FHV-AB2
into an animal cell; (b) introducing a polynucleotide sequence
encoding a candidate RNA silencing suppressor into cell; and (c)
testing for a rate or extent of FHV-AB2 RNA accumulation greater
than that for a cell not contacted with the candidate suppressor.
The method can be used to identify either silencing suppressors
from a virus or an endogenous silencing suppressor.
[0010] In another aspect, the invention provides methods for
identifying a viral RNA silencing suppressor by identifying a
polynucleotide sequence that is an overlapping gene. In some
embodiments, the gene overlaps with a RNA polymerase gene.
[0011] In yet another aspect, the invention provides methods for
identifying an inhibitor of a RNA silencing suppressor. The methods
typically comprise (a) infecting an animal cell with FHV; (b)
contacting the cell with a candidate inhibitor of a RNA silencing
suppressor; and (c) testing for a rate or extent of FHV RNA
accumulation less than that for a cell not contacted with the
candidate inhibitor. In certain embodiments, the candidate
inhibitor is a small molecule. Typically, RNA accumulation is
measured by visual assays for expressed reporter molecules and
Northern blots.
[0012] In still yet another aspect, the invention provides methods
for identifying a gene in the antiviral RNA silencing pathway of an
animal. Typically, the methods comprise: (a) providing an animal
cell expressing a polynucleotide encoding FHV-AB2; (b) inhibiting
the expression of a candidate gene in the antiviral RNA silencing
pathway; and (c) testing for a rate or extent of FHV-.DELTA.B2 RNA
accumulation greater than that for a cell where the expression of
the candidate gene in the antiviral RNA silencing pathway has not
been inhibited. In some embodiments, the polynucleotide encoding
FHV-.DELTA.B2 further comprises a reporter molecule, such as GFP.
Typically, the expression of the candidate gene is inhibited by RNA
interference, antisense DNA, or a ribozyme.
[0013] In still yet another aspect, this invention provides methods
for identifying an enhancer of the antiviral RNA silencing pathway
in an animal. The methods typically comprise (a) providing an
animal cell infected with FHV; (b) contacting the cell with a
candidate enhancer of the antiviral RNA silencing pathway; and (c)
testing for a rate or extent of FHV RNA accumulation less than that
for a cell which is not contacted with the candidate enhancer.
[0014] In another aspect, this invention provides methods for
treating or preventing a viral infection in an animal. The methods
typically comprise enhancing the antiviral RNA silencing pathway by
contacting the animal with a compound that modulates a gene
identified by the methods of this invention. In one embodiment, the
gene is AGO2 or an AGO2 homologue.
Definitions
[0015] As defined herein, the term "inactivation" refers to the act
of reducing or eliminating the expression of a particular gene.
[0016] The term "polynucleotide sequence of a virus sufficient to
activate RNA silencing" or "polynucleotide sequence of a virus that
activates RNA silencing", as used herein, refers to any portion of
the viral genome which is capable of inducing degradation of viral
or any other target RNA. Such polynucleotides typically lack
sequences encoding functional viral RNA silencing suppressors. This
is typically accomplished by deleting all or substantially all the
sequences encoding suppressors or mutating suppressor sequences to
disrupt or impair function. In certain instances, such
polynucleotides can also encode natural suppressors with weak
activity.
[0017] The term "polynucleotide sequence encoding FHV-.DELTA.B2" or
"polynucleotide encoding FHV-.DELTA.B2", as used herein, refers to
sequences that encode portions of the FHV genome sufficient to
activate RNA silencing, but that do not encode a functional B2
protein (a viral RNA silencing suppressor). This is typically
accomplished by deleting all or substantially all the sequence
encoding B2, mutating the B2 sequence to disrupt function, or
mutating the B2 sequence to reduce activity. "Polynucleotide
sequences encoding FHV-.DELTA.B2" can also encode other
polypeptides, such as reporter molecules. In certain instances,
these polynucleotides also comprise sequences which increase the
host range of FHV or sequences which facilitate introduction of FHV
into cells not normally susceptible to FHV infection.
[0018] As used herein, the term "recombinant vector" refers to a
recombinant DNA construct which has polynucleotide sequences that
enable either stable and heritable expression of the construct or
transient expression in an host. Typically, such vectors are
non-infectious and are introduced into cells via standard methods
including, but not limited to calcium phosphate-mediated
transfection, lipid-mediated transfection, electroporation, DNA
guns, etc.
[0019] As used herein, the term "heterologous" refers to any
sequence from another organism. For example, the term
"polynucleotide sequences from heterologous viruses" as used herein
refers to sequences from viruses other than the virus which
provides the sequences that activate RNA silencing.
[0020] As used herein, the term "endogenous gene" refers to any
gene which is a natural part of the genome and has not been
introduced via artificial means.
[0021] The term "oncogene" refers to any gene which is capable of
inducing neoplastic transformation. Oncogenes include, but are not
limited to, src, fos, jun, myb, abl, etc.
[0022] The term "RNA silencing" as used herein refers to the
degradation of RNA as a process induced by a natural "trigger",
e.g., viral infection, rather than artificial manipulation, which
is referred to as RNAi. In this application, the term specifically
refers to the antiviral defense mechanism by which viral RNA is
degraded in response to viral infection in a plant or animal
cell.
[0023] The term "RNAi" or "RNA interference" as used herein refers
to the degradation of RNA induced by introduction of dsRNA into a
cell or manipulations designed to induce cells to produce
artificial dsRNA.
[0024] The term "RNA silencing suppressor" as used herein refers to
any polypeptide which is capable of blocking or reducing RNA
silencing. RNA silencing suppressors in plants include p19 (tomato
bushy stunt virus), CMV 2b (cucumomosaic virus), HC-Pro
(potyviruses), P1 (rice yellow mottle sobemovirus), AC2 (African
cassaya mosaic geminovirus), and p25 (potato virus X). RNA
silencing suppressors in animals include B2 (flock house
virus).
[0025] The term "overlapping gene" as used herein refers to genes
that share common nucleotide sequence but which encode distinct
polypeptides due to different ORFs, stop points, or start points.
"Overlapping genes" are primarily found in bacteria and
viruses.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 illustrates cross-kingdom suppression of RNA
silencing in plants by an animal viral protein. The GFP-expressing
Nicotiana benthamiana leaves were co-infiltrated with a mixture of
two Agrobacterium tumefaciens strains as described (Voinnet et al.,
Cell, 103:157 (2000); Guo et al., EMBO J, 21:398 (2002)). One
directs expression of GFP and thereby induces GFP RNA silencing and
the other simultaneously expresses B2 (left leaf), 2b (right leaf)
or an untranslatable 2b coding sequence (middle leaf). The leaves
were detached and photographed under UV illumination 6 days
post-infiltration. GFP silencing is visualized in the control leaf
(middle) as a bright red color zone surrounding the infiltrated
patch due to chlorophyll fluorescence.
[0027] FIG. 2 illustrates induction and suppression of RNA
silencing in Drosophila by FHV. (A to C) A time course analysis on
the accumulation of FHV siRNAs (A) and RNAs 1-3 (B) in S2 cells
infected with FHV virions and densitometry measurements of the
accumulation levels of FHV RNA1 and siRNA are shown in (C). An RNA
marker of 22 nt in length transcribed in vitro was loaded in the
right lanes (A). (D) Accumulation of FHV RNAs in S2 cells
transfected with pRNA1 or pRNA1-.DELTA.B2 with or without dsRNA.
dsRNA corresponding to mRNA of cyclin E (cycE), GFP and two fly DCR
genes, to the 5' and 3'-terminal 1,000 nt of the AGO2 mRNA (Hammond
et al., Science, 293:1146 (2001); Bernstein et al., Nature, 409:363
(2001); Hammond et al., Nature, 404:293 (2000)), and to the
3'-terminal 500 nt of FHV RNA1, or a B2-expressing plasmid that
were co-transfected into S2 cells are indicated above each
lane.
[0028] FIG. 3 illustrates the genome organization and expression of
FHV RNA1 (Ball et al., in Virus taxonomy--Seventh report of the
international committee on taxonomy of viruses. H. V. van
Regenmortel et al., Eds. (Academic Press, 2000), pp.747). RNA1
encodes protein A, which is the catalytic subunit of the viral
RNA-dependent RNA polymerase. RNA3 encodes proteins B1 and B2. B1
is encoded in the same ORF as protein A whereas B2 is encoded in
the +1 reading frame of protein A. The initiation codon for B1
(indicated by a vertical line) is 10 nt upstream the B2 ORF. RNA2
encodes the coat protein precursor and is replicated in trans by
protein A.
[0029] FIG. 4A shows Northern blot analysis of total RNAs extracted
3 and 6 days after infiltration with 35S-GFP alone (-; lanes 1
& 6) or plus 35S-B2 (B2; lanes 2 & 7), 35S-B1 (B1; lanes 3
& 8), 35S-.DELTA.B2 (AB2; lanes 4 & 9), or 35S-T2b (T2b;
lanes 5 & 10). FIG. 4B shows the accumulation of GFP
siRNAs.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] I. Overview
[0031] This invention is based on the discovery that RNA silencing
acts as an antiviral defense mechanism in animal cells.
Specifically, this invention establishes that a virus can induce
strong viral RNA silencing and that the same viruses are equipped
with an effective silencing suppressor essential for infection.
Prior to this discovery, it was known that RNA degradation could be
artificially induced by dsRNA in animals and that RNA silencing was
an antiviral defense mechanism in plants, but it was not known that
RNA degradation could occur in response to a natural trigger, i.e,
a virus, in animals.
[0032] The discovery of a novel animal antiviral defense mechanism
offers immense opportunities for treating human and animal viral
diseases and for gene therapy. For example, viral infections can be
treated by enhancing the RNA silencing antiviral defense response,
or by blocking the action of suppressors of RNA silencing. In
addition, since RNA viruses are potent initiators of RNA silencing,
foreign sequences from endogenous human genes or heterologous
viruses can be inserted into attenuated RNA viruses to produce a
novel class of therapeutic vectors for either inactivating certain
human genes (gene therapy) or targeting other viruses in trans (as
a live attenuated vaccine).
[0033] In one aspect, this invention provides recombinant DNA
constructs comprising viral sequence sufficient to activate RNA
silencing. Such polynucleotides typically lack sequences encoding
functional viral RNA silencing suppressors. In another aspect, the
invention provides methods of identifying additional RNA silencing
suppressors. Suppressors can be identified by functional methods
using recombinant DNA constructs of this invention or by
bioinformatic/sequence analysis methods to identify other genes
with similar key features. In another aspect, this invention
provides recombinant DNA constructs for inactivating genes, wherein
the construct comprises viral sequence sufficient to activate RNA
silencing and a target gene for inactivation. In still yet another
aspect, this invention provides methods for identifying genes in
the antiviral RNA silencing pathway using recombinant DNA
constructs of this invention. This invention also provides methods
for identifying modulators of the RNA silencing suppressors and the
antiviral RNA silencing pathway, as well as methods for treating
animals infected with virus and for preventing viral infections by
upregulating the antiviral pathway.
[0034] Preparation of recombinant DNA constructs for activation of
RNA silencing is described in detail in Section II of this
application. Embodiments of the invention which use these
constructs are described in Sections III to VIII.
[0035] II. Vectors that Activate RNA Silencing
[0036] This invention provides vectors with viral polynucleotide
sequences sufficient to activate RNA silencing. These vectors have
multiple uses, including identification of RNA silencing
suppressors (Section III), gene therapy (Section IV), and
identification of genes in the antiviral RNA silencing defense
pathway (Section V).
[0037] These vectors typically lack sequences encoding functional
viral RNA silencing suppressors. This is typically accomplished by
deleting all or substantially all the sequences encoding
suppressors, mutating suppressor sequences to disrupt function, or
mutating suppressor sequences to reduce activity. In certain
instances, the polynucleotides can also encode natural suppressors
with weak activity.
[0038] One of skill will recognize that the viral polynucleotide
sequence can be from any virus capable of inducing RNA silencing.
In one embodiment, the vector used for gene inactivation comprises
polynucleotide portions of FHV sufficient to activate RNA
silencing.
[0039] In certain embodiments, the vectors are infectious viral
vectors. Such vectors comprise a viral genome and the target gene.
Typically, the viral genome has been modified to remove sequences
that confer virulence.
[0040] Infectious viral vectors of this invention are typically
capable of infecting a broad range of hosts including humans, dogs,
cats, horses, cows, monkey, etc.; usually, these viral vectors are
capable of efficiently infecting all humans. Any viruses that have
been developed for use as gene therapy vectors can be used.
Exemplary viruses include retroviruses (including lentiviruses),
adenoviruses, adeno-associated viruses, herpes simplex virus type
1, etc. Additionally, viral vectors can be derived from the genome
of human or bovine adenoviruses, vaccinia virus, herpes virus,
minute virus of mice (MVM), HIV, sindbis virus, Rous sarcoma virus,
and MoMLV. In certain embodiments, the infectious viral vectors of
this invention further comprise polynucleotide sequences that alter
the host range. Infectious viral vectors with a host range
different from or broader than that of the native viral
polynucleotide sequence can be constructed by incorporating these
determinants of host range.
[0041] In another embodiment, the invention provides non-infectious
recombinant vectors (amplicons) which can be introduced into a cell
to produce a stable and heritable phenotype. For example, an
amplicon can comprise a promoter and terminator which directs
transcription of modified viral vector RNA. The modifications can
include deletion or mutation of viral genes required for spread of
the virus or any other functions that are secondary to
replication.
[0042] In certain embodiments, these vectors can have inverted
repeats in the transcribed regions. In yet another embodiment, the
invention provides non-infectious transgenic vectors that are only
transiently expressed in the host cell.
[0043] Non-infectious vectors can be introduced into cells using
standard methods known to those of skill in the art. Such methods
include electroporation, the use of DNA guns, calcium-phosphate
mediated transfection, lipid-mediated transfection, the use of kits
designed for such purposes, and the like. The vectors can also be
introduced via other systems including, but not limited, to an HVJ
(Sendai virus)-liposome gene delivery system (see, e.g., Kaneda et
al., Ann. N.Y. Acad. Sci. 811:299-308 (1997)); a "peptide vector"
(see, e.g., Vidal et al., CR Acad. Sci III 32:279-287 (1997)); as a
gene in an episomal or plasmid vector (see, e.g., Cooper et al.,
Proc. Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum
Gene Ther. 8:575-584 (1997)); as a gene in a peptide-DNA aggregate
(see, e.g., Niidome et al., J. Biol. Chem. 272:15307-15312 (1997));
as "naked DNA" (see, e.g., U.S. Pat. No. 5,580,859 and U.S. Pat.
No. 5,589,466); in lipidic vector systems (see, e.g., Lee et al.,
Crit Rev Ther Drug Carrier Syst. 14:173-206 (1997)); polymer coated
liposomes (Marin et al., U.S. Pat. No. 5,213,804, issued May 25,
1993; Woodle et al., U.S. Pat. No. 5,013,556, issued May 7, 1991);
cationic liposomes (Epand et al., U.S. Pat. No. 5,283,185, issued
Feb. 1, 1994; Jessee, J. A., U.S. Pat. No. 5,578,475, issued Nov.
26, 1996; Rose et al, U.S. Pat. No. 5,279,833, issued Jan. 18,
1994;
[0044] Portions of a virus sufficient to activate RNA silencing can
easily be identified using standard mutagenesis and deletion
methods known to those of skill in the art. For example, a vector
comprising a viral genome with various deletions and lacking
functional RNA silencing suppressors, can be transfected into cells
and tested for RNA silencing activity. Assays for RNA silencing
include measuring accumulation of viral RNA or detecting
intermediates produced during RNA degradation, e.g., siRNA, etc.
Methods for detecting and quantifying RNA are described in Section
VI, Part B, below.
[0045] Vectors of the present invention can be constructed using
standard methods in molecular biology, such as those described in
many general molecular biology textbooks such as Sambrook et al.,
Molecular Cloning a Laboratory Manual 2nd Ed. Cold Spring Harbor
Press, Cold Spring Harbor (1989) and Ausubel et al., Current
Protocols in Molecular Biology, (current edition).
[0046] III. Identification of RNA Silencing Suppressors
[0047] In one aspect, this invention provides RNA silencing
suppressors of animal viruses. These animal viruses can be DNA or
RNA viruses. A particular virus may encode more than one RNA
silencing suppressor.
[0048] In one embodiment, the invention provides a RNA silencing
suppressor for FHV called B2. In other embodiments, this invention
provides RNA silencing suppressors for other animal viruses,
including those viruses where infection is widespread, has severe
effects, or where there has been limited success in developing
effective vaccines or therapeutics. Exemplary viruses include HIV,
HCV, HBV, influenza viruses, measles virus etc. In certain
embodiments, the present invention also provides methods of
identifying endogenous/cellular RNA silencing suppressors (see,
Anandalakshmi et al., Science, 290:142-144 (2000)).
[0049] Silencing suppressors can be identified using the teachings
provided here and standard methods known to those of skill in the
art. For example, suppressors are identified by sequence analysis,
functional tests using vectors described in Section II, or
combinations herewith.
[0050] A. Sequence Analysis
[0051] A majority of known plant viral RNA silencing suppressors
are "overlapping" genes (see, Table 1).
1TABLE I Suppressor Overlapped Virus name Genome type gene gene
Reference Cucumber mosaic Plus-strand 2b RNA- Brigneti et al. virus
RNA dependent EMBO J 17:6739- RNA 6746 (1998) polymerase (RdRP)
Tomato aspermy virus Plus-strand 2b RdRP Li et al. EMBO J RNA
18:2683-2691 (1999) Tomato bushy stunt Plus-strand p19 Movement
Voinnet et virus RNA protein (p23) al.PNAS USA 96:14147-14152
(1999) Africa cassava mosaic Single-stranded AC2 Replication-
Voinnet et virus circular DNA associated al.PNAS USA protein
96:14147-14152 (AC1) (1999) Turnip yellow mosaic Plus-strand OP
RdRP Ding SW, virus RNA unpublished data Potato virus X RNA p25
p12
[0052] For example, the RNA silencing suppressor for the cucumber
mosaic virus is an overlapping gene in the +1 frame of the viral
RNA-dependent RNA polymerase (RdRP) gene. Similarly, the viral RNA
silencing suppressor for FHV is also an overlapping gene in the +1
frame of the viral RNA polymerase gene.
[0053] Thus analysis of viral genomes can be used to identify RNA
silencing suppressors. Viral genomes are thus conveniently examined
for "overlapping" genes. Overlapping genes are often conserved only
in a specific taxonomic grouping and their encoded proteins are
likely to have unusual biochemical properties. In some embodiments,
particular attention is paid to "overlapping" genes of RNA
polymerase in the +1 reading frame. However, it will be readily
appreciated by those of skill in the art that putative viral RNA
silencing suppressors are not limited to this particular
embodiment; the suppressors can overlap with any gene in any
reading frame.
[0054] In some embodiments, viral genomes will be examined for
genes that appear to be required for virulence and virus
accumulation, but for which there is no known specific function.
Viral genomes are also typically examined for genes that are
essential for viral infection in certain cell types but not
essential in other cell types. Genes with these characteristics are
good candidates for suppressor genes.
[0055] As an example, the gene F has been identified as a RNA
silencing suppressor for HCV based on sequence analysis. The F
protein is a novel protein synthesized from the initiation codon of
the HCV polyprotein followed by a ribosomal frameshift into the
-2/+1 reading frame (see, SEQ ID NO:1-2; Xu et al. EMBO J 20:3840
(2001)). Table 2 lists other RNA silencing suppressors for various
viruses.
2TABLE 2 Genome Overlap- Virus name type Suppressor ped gene
Reference HIV-1 and HIV-2 RNA tat, rev env Keese & Gibbs PNAS
89:9489-9493 (1992) HIV-1 RNA vpu env Keese & Gibbs PNAS
89:9489-9493 (1992) HIV-2 RNA vpx vif Keese & Gibbs PNAS
89:9489-9493 (1992) Measles virus RNA C and V P Fields Virology,
Fourth Edition. 2001 Chpt 44 Influenza virus B RNA NB NA Fields
Virology, Fourth Edition. 2001 Chpt 46 Influenza virus B RNA BM2 M1
Fields Virology, Fourth Edition. 2001 Chpt 46 Influenza virus A/B/C
RNA NS1/NS2 NS1/NS2 Fields Virology, Fourth Edition. 2001 Chpt 46
Papillomaviruses DNA E4 E2 Fields Virology, Fourth Edition. 2001
Chpt 65 Hepadnaviruses (includes DNA X P Fields Virology, Fourth
Edition. 2001 Hepatitis B virus) Chpt 86 Hepatitis C virus RNA F C
Xu et al. EMBO J 20:3840 (2001)
[0056] Once putative RNA silencing suppressors are identified, they
can be tested using any functional test known to those of skill in
the art, such as those described in the following section.
[0057] B. Functional Analysis
[0058] RNA silencing suppressors can also be identified via
functional tests using vectors described in Section II. Typically,
a test will examine the ability of a polypeptide encoded by a
candidate RNA silencing suppressor gene to hinder, block, or slow
RNA silencing induced by the viral vector. RNA levels in a cell can
be measured using methods described in Section VI, Part B,
below.
[0059] In certain embodiments, the method comprises expressing a
polynucleotide sequence of a virus sufficient to activate RNA
silencing as defined herein ("silenced viral sequence") in a cell,
introducing a polynucleotide encoding a candidate RNA silencing
suppressor into the cell, and testing for increased rate or extent
of accumulation of the "silenced viral sequence".
[0060] It will be appreciated that the polynucleotide sequence can
be part of either an infectious vector or a non-infectious vector.
It will further be appreciated that the "silenced viral sequence"
and candidate suppressor sequence can either be on the same or
different vector and introduced at varying times. In some
embodiments, the "silenced viral sequence" is introduced before the
candidate suppressor gene. Based on studies with plant suppressors,
it is known that certain viral RNA silencing suppressors target
early stages of RNA silencing, while others target later stages
(see, Li and Ding. Viral suppressors of RNA silencing. Curr. Opin.
Biotech., 12:150-154 (2001). Suppressors which target early stages
of the RNA silencing pathway are unlikely to be active unless
expressed before or during the initiation of RNA silencing.
Therefore, in some embodiments, the suppressor gene is either
introduced before or during RNA silencing-either on the same vector
as the "silenced viral sequence", on a separate vector prior to
introduction of the "silenced viral sequence", or on the same
vector as the "silenced viral sequence" but engineered to be
expressed first.
[0061] The "silenced viral sequence" can be expressed in any animal
cell where the antiviral RNA silencing pathway is activated in
response to the "silenced viral sequence". In certain embodiments,
Drosophila cells are used.
[0062] Candidate RNA silencing suppressors can be any gene
identified by sequence analysis described in the above section or
any other gene which has properties or a sequence which suggest
that the gene may be a RNA silencing suppressor. For identification
of viral RNA silencing suppressors, the candidate gene can be from
a virus. For identification of endogenous suppressors, the
candidate gene can be from the genome of the same organism as the
host cell, or from the genome of a different organism.
[0063] RNA accumulation of the "silenced viral sequence" can be
measured using any method known to those of skill in the art, such
as those described in Section VI, Part B; these methods include
Northern blot or assays to detect reporter molecules linked to the
polynucleotide. The reporter molecules can either be detectable
fluorescence molecules or selectable antibiotic markers. In one
embodiment, RNA accumulation is detected using an where the first
polynucleotide is coupled to GFP. Suppression of RNA silencing
allows expression of GFP, which can be visualized by UV
illumination. Active RNA silencing generates a red fluorescent
zone.
[0064] In one embodiment, the "silenced viral sequence" encodes
FHV-.DELTA.B2. It has been demonstrated that a vector encoding such
a sequence is deficient in silencing suppression and does not
accumulate to detectable levels in Drosophila cells; however, this
defect can be rescued in trans by co-transfection with a plasmid
expressing B2.
[0065] IV. Gene Therapy & Vaccination Against Pathogens
[0066] In certain embodiments, the above-described vectors for
inducing RNA silencing (Section II) further comprise a target gene,
wherein inactivation of the target gene is desired. Those of skill
in the art will recognize that vectors of this invention can be
used for any application where a reduction in mRNA expression
levels of specific genes or the protein expression levels of
specific genes is desired. The vectors of this invention are
particularly useful for methods where targeted inactivation of
closely related multigene families is desired. Any gene that can be
targeted with antisense technology can also be targeted with this
invention.
[0067] In certain embodiments, the target genes for the vectors of
this invention are portions of heterologous pathogen genomes
wherein inactivation of such portions is sufficient to reduce or
eliminate infection by the pathogen. In a typical embodiment, the
pathogen will be a virus. By activating sustained RNA degradation
of these viral genomes, these vectors are expected to be useful for
both the treatment and prevention of viral infections in
animals.
[0068] In other embodiments, the target genes for the vectors are
endogenous genes. Typically, the endogenous genes are ones where
the expression or overexpression of such genes induces a disease or
a medical condition. In some embodiments, the targeted endogenous
genes encode polypeptides that contribute to cancer, such as
oncogenes, e.g., src, abl, ras, etc. and growth factors, such as
the epidermal growth factor. In other embodiments, the targeted
endogenous gene encodes a polypeptide that promotes abnormal
angiogenesis, a factor that contributes to tumor growth and ocular
diseases (e.g., diabetic retinopathy and macular degeneration).
Such angiogenesis-promoting polypeptides include vascular
endothelial growth factor (VEGF). In still other embodiments, the
vectors can be used to treat any monogenic autosomal dominant
disease (e.g., familial hypercholesteremia, dominant forms of
retinal degeneration, Huntington's disease, myotonic dystrophy,
hemophilia, etc.). In contrast to autosomal recessive diseases,
treatment of these diseases requires that an aberrant gene is
silenced (see, Gerard and Collen, Cardiovascular Research
35:451-458 (2001)).
[0069] In other embodiments, the endogenous genes are ones where it
is desirable to understand the precise function of the genes or the
effect of inactivating the gene. Such vectors are valuable tools in
functional genomic screening assays.
[0070] In still other embodiments, the endogenous genes are ones
where inactivation the genes can be used to generate animal models
for disease. Typically, these diseases are monogenic diseases where
deletion of a single gene or mutation to generate a nonfunctional
gene is sufficient to induce a disease phenotype (i.e., monogenic
disease), such as cystic fibrosis, Duchenne muscular dystrophy,
hemophilia, ADA deficiency, and familial hypercholesteremia. For
example, a vector which activates silencing of the CFTR genes can
be used to generate an animal model for cystic fibrosis.
[0071] V. Methods for Identifying Genes in the Antiviral RNA
Silencing Pathway
[0072] This invention further provides methods for identifying
genes in the antiviral RNA silencing pathway using vectors
described in Section II. By identifying viral RNA silencing
suppressors, this invention provides vectors which can be used as
tools to determine whether a particular gene is part of the
antiviral defense pathway. These "reporter vectors" can be any
vector expressing viral polynucleotide sequences sufficient to
activate RNA silencing as defined herein, i.e., any vector which is
rapidly degraded after expression. Genes in the antiviral silencing
pathway are identified by inhibiting the expression of a candidate
gene in cells expressing the "reporter vector" and looking for an
increased rate or extent of viral RNA accumulation.
[0073] In some embodiments, the "reporter vector" comprises
portions of the FHV genome sufficient to activate RNA silencing,
lacking a functional RNA silencing suppressor, and linked to a
reporter molecule. The vector can also comprise additional
polynucleotide sequences which increase the host range of FHV or
sequences which facilitate introduction of the vector into cells.
In one embodiment, the "reporter vector" encodes FHV-.DELTA.B2
linked to a reporter molecule, such as GFP.
[0074] The above-described "reporter vectors" can be engineered to
be expressed in any animal cell where it is capable of activating
an antiviral RNA silencing pathway. The "reporter vector" can be
used to identify components of the antiviral pathway for the
particular type of animal that the cell is derived from. For
example, introduction of the "reporter vector" into a Drosophila
cell will allow identification of fly antiviral RNA silencing
pathway genes. Sequence analysis can be used to identify homologues
in other organisms.
[0075] Expression of a candidate gene in the antiviral defense
pathway can be inhibited using any standard method known to those
of skill in the art, such as RNA interference, antisense molecules
and ribozymes. Accumulation of the above-described "reporter
vectors" can be measured using standard methods for detection of
RNA. In certain embodiments, the vectors are coupled to a tag or
label which allows visual detection of the expression product, such
as GFP.
[0076] This invention further comprises any novel genes identified
by such a method.
[0077] VI. Methods for Identifying Modulators of RNA Silencing
Suppressors and Modulators of Genes in the Antiviral RNA Silencing
Pathway
[0078] In certain embodiments, the invention comprises methods for
identifying modulators of RNA silencing suppressors and modulators
of the antiviral RNA silencing pathway. Typically, the methods of
this invention are used to identify inhibitors of RNA silencing
suppressors and enhancers of the antiviral RNA silencing pathway.
The term "test compound" or "drug candidate" or "modulator" or
grammatical equivalents as used herein describe any molecule, e.g.,
protein, oligopeptide, small organic molecule, polysaccharide,
polynucleotide, etc., to be tested for the capacity to directly or
indirectly alter the activity of RNA silencing suppressors and/or
genes in the antiviral RNA silencing pathway.
[0079] In some embodiments, an animal cell or animal is infected
with virus and the animal cell is contacted with a candidate
modulator or the animal is "administered" the modulator. By
"administration" or "contacting" herein is meant that the candidate
agent is administered in such a manner as to allow the agent to act
upon the animal. Generally, a plurality of different modulator
concentrations are tested to obtain a differential response to the
various concentrations. Typically, one of these concentrations
serves as a negative control, i.e., at zero concentration or below
the level of detection.
[0080] A. Compounds & Biomolecules to be Screened
[0081] The compounds tested as modulators of the activity of RNA
silencing suppressors and the antiviral RNA silencing pathway can
be any small chemical compound, or a biological entity, such as a
protein, sugar, nucleic acid or lipid. Alternatively, modulators
can be genetically altered versions of the genes. Typically, test
compounds will be small chemical molecules and peptides.
Essentially any chemical compound can be used as a potential
modulator or ligand in the assays of the invention, although most
often compounds can be dissolved in aqueous or organic (especially
DMSO-based) solutions are used. It will be appreciated that there
are many suppliers of chemical compounds, including Sigma (St.
Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis,
Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and
the like.
1. Combinatorial Chemistry Libraries
[0082] In certain embodiments, combinatorial libraries of potential
modulators will be screened for an ability to bind to modulate RNA
silencing suppressors and components of the antiviral RNA silencing
defense pathway. Conventionally, new chemical entities with useful
properties are generated by identifying a chemical compound (called
a "lead compound") with some desirable property or activity, e.g.,
inhibiting activity, creating variants of the lead compound, and
evaluating the property and activity of those variant
compounds.
[0083] In one embodiment, the drug screening method involves
providing a combinatorial chemical or peptide library containing a
large number of potential therapeutic compounds (potential
modulator or ligand compounds). Such "combinatorial chemical
libraries" or "ligand libraries" are then screened in one or more
assays, as described herein, to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
[0084] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0085] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature,
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication No. WO
93/20242), random bio-oligomers (e.g., PCT Publication No. WO
92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),
diversomers such as hydantoins, benzodiazepines and dipeptides
(Hobbs et al., Proc. Nat. Acad. Sci. USA, 90:6909-6913 (1993)),
vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.,
114:6568 (1992)), nonpeptidal peptidomimetics with glucose
scaffolding (Hirschmann et al., J. Amer. Chem. Soc., 114:9217-9218
(1992)), analogous organic syntheses of small compound libraries
(Chen et al., J. Amer. Chem. Soc., 116:2661 (1994)),
oligocarbamates (Cho et al., Science, 261:1303 (1993)), and/or
peptidyl phosphonates (Campbell et al., J. Org. Chem., 59:658
(1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook,
all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat.
No. 5,539,083), antibody libraries (see, e.g., Vaughn et al.,
Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287),
carbohydrate libraries (see, e.g., Liang et al., Science,
274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic
molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan
18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;
thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;
pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino
compounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514, and
the like).
[0086] A number of well known robotic systems have also been
developed for solution phase chemistries. These systems include
automated workstations like the automated synthesis apparatus
developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and
many robotic systems utilizing robotic arms (Zymate II, Zymark
Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto,
Calif.), which mimic the manual synthetic operations performed by a
chemist. The above devices, with appropriate modification, are
suitable for use with the present invention. In addition, numerous
combinatorial libraries are themselves commercially available (see,
e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,
St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals,
Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
2. Proteins and Nucleic Acids as Potential Modulators
[0087] In one embodiment, the modulators are proteins, often
naturally occurring proteins or fragments of naturally occurring
proteins. Thus, e.g., cellular extracts containing proteins, or
random or directed digests of proteinaceous cellular extracts, may
be used. In this way libraries of proteins may be made for
screening in the methods of the invention. These can include, but
are not limited to, libraries of bacterial, fungal, viral, and
mammalian proteins. Typically, the libraries comprise human
proteins.
[0088] In one embodiment, modulators are peptides of from about 5
to about 30 amino acids, from about 5 to about 20 amino acids, or
from about 7 to about 15. The peptides may be digests of naturally
occurring proteins as is outlined above, random peptides, or
"biased" random peptides. By "randomized" or grammatical
equivalents herein is meant that the nucleic acid or peptide
consists of essentially random sequences of nucleotides and amino
acids, respectively. Since these random peptides (or nucleic acids,
discussed below) are often chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized candidate bioactive proteinaceous
agents.
[0089] In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. Typically, the
library is biased. That is, some positions within the sequence are
either held constant, or are selected from a limited number of
possibilities. In another embodiment, the nucleotides or amino acid
residues are randomized within a defined class, e.g., of
hydrophobic amino acids, hydrophilic residues, sterically biased
(either small or large) residues, towards the creation of nucleic
acid binding domains, the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, etc.
[0090] Modulators can also be nucleic acids, as defined above. As
described above generally for proteins, nucleic acid modulating
agents may be naturally occurring nucleic acids, random nucleic
acids, or "biased" random nucleic acids. Digests of prokaryotic or
eukaryotic genomes may be used as is outlined above for
proteins.
[0091] B. The Screening Process
[0092] Candidate modulators can be identified by infecting animal
cells with any native virus which activates RNA silencing. The
effect of such modulators can then be determined by measuring
accumulation of viral RNA. RNA levels can be determined using any
standard method known to those of skill in the art, such as visual
assays indicating transcription of reporter molecules or Northern
blots. In other embodiments, RNA levels can be measured using
labeled probes or amplification-based assays.
[0093] Probes to detect RNA can be a nucleotide/deoxynucleotide
probe that is complementary to and hybridizes with the RNA and
includes, but is not limited to, oligonucleotides, cDNA or RNA.
Probes also should contain a detectable label as defined in the
art. In one method the RNA is detected after immobilizing the RNA
to be examined on a solid support such as nylon membranes and
hybridizing the probe with the sample. Following washing to remove
the non-specifically bound probe, the label is detected. In another
method detection of the RNA is performed in situ. In this method
permeabilized cells or tissue samples are contacted with a
detectably labeled nucleic acid probe for sufficient time to allow
the probe to hybridize with the target RNA. Following washing to
remove the non-specifically bound probe, the label is detected. For
example a digoxygenin labeled riboprobe (RNA probe) that is
complementary to the RNA is detected by binding the digoxygenin
with an anti-digoxygenin secondary antibody and developed with
nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.
[0094] Often, amplification-based assays are performed to measure
the expression level of viral RNAs. These assays are typically
performed in conjunction with reverse transcription. In such
assays, a nucleic acid sequence acts as a template in an
amplification reaction (e.g., Polymerase Chain Reaction, or PCR).
In a quantitative amplification, the amount of amplification
product will be proportional to the amount of template in the
original sample. Comparison to appropriate controls provides a
measure of the amount of RNA. Methods of quantitative amplification
are well known to those of skill in the art. Detailed protocols for
quantitative PCR are provided, e.g., in Innis et al., PCR
Protocols, A Guide to Methods and Applications (1990).
[0095] In some embodiments, a TaqMan based assay is used to measure
expression. TaqMan based assays use a fluorogenic oligonucleotide
probe that contains a 5' fluorescent dye and a 3' quenching agent.
The probe hybridizes to a PCR product, but cannot itself be
extended due to a blocking agent at the 3' end. When the PCR
product is amplified in subsequent cycles, the 5' nuclease activity
of the polymerase, e.g., AmpliTaq, results in the cleavage of the
TaqMan probe. This cleavage separates the 5' fluorescent dye and
the 3' quenching agent, thereby resulting in an increase in
fluorescence as a function of amplification (see, e.g., literature
provided by Perkin-Elmer, e.g., www2.perkin-elmer.com).
[0096] Other suitable amplification methods include, but are not
limited to, ligase chain reaction (LCR) (see Wu & Wallace,
Genomics, 4:560 (1989), Landegren et al., Science, 241:1077 (1988),
and Barringer et al., Gene, 89:117 (1990)), transcription
amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173
(1989)), self-sustained sequence replication (Guatelli et al.,
Proc. Nat. Acad. Sci. USA, 87:1874 (1990)), dot PCR, and linker
adapter PCR, etc.
[0097] VII. Therapeutic and Prophylactic Applications
[0098] This invention also provides methods for treating or
preventing viral infection by upregulating degradation of viral RNA
and thus reducing virus levels. Typically, degradation of viral RNA
is upregulated by either activating the antiviral RNA silencing
pathway or inhibiting any RNA silencing suppressors using
modulators identified with methods of this invention.
[0099] In one embodiment, the antiviral silencing pathway is
activated by administering a pharmaceutical composition that either
upregulates the expression level of a gene in the pathway or
enhances of the activity of a gene in the pathway. The gene can be
one identified by the methods of this invention. In one embodiment,
the activity or expression level of AGO2 or an AGO2 homologue is
increased.
[0100] In another embodiment, the suppression of RNA silencing is
blocked or reduced by administering a pharmaceutical composition
that either inhibits the activity of a RNA silencing suppressor or
reduces the expression level of a RNA silencing suppressor.
Compounds with these particular attributes can be identified using
methods of this invention. Pharmaceutical compositions, dosages,
and administration modes are described below.
[0101] VIII. Compositions Comprising Modulators Identified in this
Invention or Gene Therapy Vectors & Pharmaceutical
Administration of such Compositions
[0102] A. Dosage
[0103] In one embodiment, a therapeutically effective dose of a
gene therapy vector or a modulator of a RNA silencing suppressor or
gene in the antiviral RNA silencing pathway is administered to a
patient. By "therapeutically effective dose" herein is meant a dose
that produces effects for which it is administered. The exact dose
will depend on the purpose of the treatment, and will be
ascertainable by one skilled in the art using known techniques
(e.g., Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery;
Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992), Dekker,
ISBN 0824770846, 082476918X, 0824712692, 0824716981; Lloyd, The
Art, Science and Technology of Pharmaceutical Compounding (1999);
and Pickar, Dosage Calculations (1999)). As is known in the art,
adjustments for systemic versus localized delivery, and rate of new
protease synthesis, as well as the age, body weight, general
health, sex, diet, time of administration, drug interaction and the
severity of the condition may be necessary, and will be
ascertainable with routine experimentation by those skilled in the
art.
[0104] A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals. Thus
the methods are applicable to both human therapy and veterinary
applications. Typically, the patient is a mammal and usually the
patient is human.
[0105] B. Administration & Pharmaceutical Compositions
[0106] The administration of the gene therapy vectors or modulators
of the present invention can be done in a variety of ways as
discussed above, including, but not limited to, orally,
subcutaneously, intravenously, intranasally, transdermally,
intraperitoneally, intramuscularly, intrapulmonary, vaginally,
rectally, intraocularly, or directly applied as a solution or
spray.
[0107] The pharmaceutical compositions of the present invention
comprise modulators in a form suitable for administration to a
patient. In one embodiment, the pharmaceutical compositions are in
a water-soluble form, such as being present as pharmaceutically
acceptable salts, which is meant to include both acid and base
addition salts. "Pharmaceutically acceptable acid addition salt"
refers to those salts that retain the biological effectiveness of
the free bases and that are not biologically or otherwise
undesirable, formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and
the like, and organic acids such as acetic acid, propionic acid,
glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic
acid, succinic acid, fumaric acid, tartaric acid, citric acid,
benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the
like. "Pharmaceutically acceptable base addition salts" include
those derived from inorganic bases such as sodium, potassium,
lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum salts and the like. Salts derived from
pharmaceutically acceptable organic non-toxic bases include salts
of primary, secondary, and tertiary amines, substituted amines
including naturally occurring substituted amines, cyclic amines and
basic ion exchange resins, such as isopropylamine, trimethylamine,
diethylamine, triethylamine, tripropylamine, and ethanolamine.
[0108] The pharmaceutical compositions may also include one or more
of the following: carrier proteins such as serum albumin; buffers;
fillers such as microcrystalline cellulose, lactose, corn and other
starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol.
[0109] The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. For example, unit dosage forms suitable for oral
administration include, but are not limited to, powder, tablets,
pills, capsules and lozenges. It is recognized that protein
modulators (e.g., antibodies, gene therapy constructs, ribozymes,
small organic molecules, etc.) when administered orally, should be
protected from digestion. It is also recognized that, after
delivery to other sites in the body (e.g., circulatory system,
lymphatic system, or the tumor site) the modulators of the
invention may need to be protected from excretion, hydrolysis,
proteolytic digestion or modification, or detoxification by the
liver. In all these cases, protection is typically accomplished
either by complexing the molecule(s) with a composition to render
it resistant to acidic and enzymatic hydrolysis, or by packaging
the molecule(s) in an appropriately resistant carrier, such as a
liposome or a protection barrier or by modifying the molecular
size, weight, and/or charge of the modulator. Means of protecting
agents from digestion degradation, and excretion are well known in
the art.
[0110] The compositions for administration will commonly comprise a
gene therapy vector or modulator dissolved in a pharmaceutically
acceptable carrier, typically, an aqueous carrier. A variety of
aqueous carriers can be used, e.g., buffered saline and the like.
These solutions are sterile and generally free of undesirable
matter. These compositions may be sterilized by conventional, well
known sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, e.g.,
sodium acetate, sodium chloride, potassium chloride, calcium
chloride, sodium lactate and the like. The concentration of active
agent in these formulations can vary widely, and will be selected
primarily based on fluid volumes, viscosities, body weight and the
like in accordance with the particular mode of administration
selected and the patient's needs (e.g., Remington's Pharmaceutical
Science (15th ed., 1980) and Goodman & Gillman, The
Pharmacologial Basis of Therapeutics (Hardman et al., eds.,
1996)).
[0111] Thus, a typical pharmaceutical composition for intravenous
administration would be about 0.1 to 10 mg per patient per day.
Dosages from 0.1 up to about 100 mg per patient per day may be
used, particularly when the drug is administered to a secluded site
and not into the blood stream, such as into a body cavity or into a
lumen of an organ. Substantially higher dosages are possible in
topical administration. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art, e.g., Remington's Pharmaceutical Science and
Goodman and Gillman, The Pharmacologial Basis of Therapeutics,
supra.
[0112] The compositions containing gene therapy vectors or
modulators can be administered for therapeutic or prophylactic
treatments to treat the disease or conditions being treated, such
as a viral infection. In therapeutic applications, compositions are
administered to a patient suffering from a disease in an amount
sufficient to cure or at least partially arrest the disease and its
complications. An amount adequate to accomplish this is defined as
a "therapeutically effective dose." Amounts effective for this use
will depend upon the severity of the disease and the general state
of the patient's health. Single or multiple administrations of the
compositions may be administered depending on the dosage and
frequency as required and tolerated by the patient. In any event,
the composition should provide a sufficient quantity of the agents
of this invention to effectively treat the patient. An amount of
modulator that is capable of preventing or slowing the development
of a disease in a mammal is referred to as a "prophylactically
effective dose." The particular dose required for a prophylactic
treatment will depend upon the medical condition and history of the
mammal, the particular disease being prevented, as well as other
factors such as age, weight, gender, administration route,
efficiency, etc. Such prophylactic treatments may be used, e.g., in
a mammal who has previously had a disease to prevent a recurrence
of the disease, or in a mammal who is expected to be susceptible to
such a disease.
[0113] It will be appreciated that the present modulating compounds
can be administered alone or in combination with additional
modulating compounds or with other therapeutic agent for treatment
of the particular disease or condition, e.g., for treatment of
viral infection, the composition can be administered together with
other agents or treatments that enhance the immune response to
viral infection.
[0114] C. Issues Specific to Adminstration and Formulation of Gene
Therapy Constructs
[0115] In some embodiments of the invention, gene therapy
constructs are conjugated to a cell receptor ligand for facilitated
uptake (e.g., invagination of coated pits and internalization of
the endosome) through an appropriate linking moiety, such as a DNA
linking moiety (Wu et al., J. Biol. Chem. 263:14621-14624 (1988);
WO 92/06180). For example, gene constructs can be linked through a
polylysine moiety to asialo-oromucocid, which is a ligand for the
asialoglycoprotein receptor of hepatocytes.
[0116] Similarly, viral envelopes used for packaging gene
constructs can be modified by the addition of receptor ligands or
antibodies specific for a receptor to permit receptor-mediated
endocytosis into specific cells (see, e.g., WO 93/20221, WO
93/14188, WO 94/06923). In some embodiments of the invention, the
DNA constructs of the invention are linked to viral proteins, such
as adenovirus particles, to facilitate endocytosis (Curiel et al.,
Proc. Natl. Acad. Sci. U.S.A. 88: 8850-8854 (1991)). In other
embodiments, molecular conjugates of the instant invention can
include microtubule inhibitors (WO/9406922); synthetic peptides
mimicking influenza virus hemagglutinin (Plank et al., J. Biol.
Chem. 269:12918-12924 (1994)); and nuclear localization signals
such as SV40 T antigen (WO93/19768).
[0117] When used for pharmaceutical purposes, the formulations of
the invention include a buffer that can contain a
delivery-enhancing compound. The buffer can be any pharmaceutically
acceptable buffer, such as phosphate buffered saline or sodium
phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile
water, and other buffers known to the ordinarily skilled artisan
such as those described by Good et al. (1966) Biochemistry 5:467.
The pH of the buffer in the pharmaceutical composition comprising a
gene therapy construct, for example, is typically in the range of
6.4 to 8.4, 7 to 7.5, or 7.2 to 7.4.
[0118] The compositions of this invention can additionally include
a stabilizer, enhancer or other pharmaceutically acceptable
carriers or vehicles. A pharmaceutically acceptable carrier can
contain a physiologically acceptable compound that acts, for
example, to stabilize the gene therapy construct. A physiologically
acceptable compound can include, for example, carbohydrates, such
as glucose, sucrose or dextrans, antioxidants, such as ascorbic
acid or glutathione, chelating agents, low molecular weight
proteins or other stabilizers or excipients. Other physiologically
acceptable compounds include wetting agents, emulsifying agents,
dispersing agents or preservatives, which are particularly useful
for preventing the growth or action of microorganisms. Various
preservatives are well known and include, for example, phenol and
ascorbic acid. One skilled in the art would know that the choice of
pharmaceutically acceptable carrier depends on the route of
administration and the particular physio-chemical characteristics
of the gene therapy vector. Examples of carriers, stabilizers or
adjuvants can be found in Martin, Remington's Pharm.Sci., 15th Ed.
(Mack Publ. Co., Easton, Pa. 1975), which is incorporated herein by
reference.
[0119] The gene therapy vector can be delivered to any tissue or
organ, including neoplastic tissues such as cancer tissue, using
any delivery method known to the ordinarily skilled artisan for
example, intratumoral or intravesical administration. Tissues and
organs include any tissue or organ having an epithelial membrane
such as the gastrointestinal tract, the bladder, respiratory tract,
and the lung. Examples include but are not limited to carcinoma of
the bladder and upper respiratory tract, vulva, cervix, vagina or
bronchi; local metastatic tumors of the peritoneum;
broncho-alveolar carcinoma; pleural metastatic carcinoma; carcinoma
of the mouth and tonsils; carcinoma of the nasopharynx, nose,
larynx, oesophagus, stomach, colon and rectum, gallbladder, or
skin; or melanoma.
[0120] In some embodiments of the invention, the therapeutic agent
is formulated in mucosal, topical, and/or buccal formulations,
particularly mucoadhesive gel and topical gel formulations.
Exemplary permeation enhancing compositions, polymer matrices, and
mucoadhesive gel preparations for transdermal delivery are
disclosed in U.S. Pat. No. 5,346,701. Such formulations are
especially useful for the treatment of cancers of the mouth, head
and neck cancers (e.g., cancers of the tracheobronchial epithelium)
skin cancers (e.g., melanoma, basal and squamous cell carcinomas),
cancers of the intestinal mucosa, vaginal mucosa, and cervical
cancer.
[0121] The formulations of the invention are typically administered
to enhance transfer of an agent to a cell. The cell can be provided
as part of a tissue, such as an epithelial membrane, or as an
isolated cell, such as in tissue culture. The cell can be provided
in vivo, ex vivo, or in vitro. In some embodiments of the
invention, the compositions of the invention are administered ex
vivo to cells or tissues explanted from a patient, then returned to
the patient. Examples of ex vivo administration of therapeutic gene
constructs include Arteaga et al., Cancer Research 56(5):1098-1103
(1996); Nolta et al., Proc Natl. Acad. Sci. USA 93(6):2414-9
(1996); Koc et al., Seminars in Oncology 23 (1):46-65 (1996); Raper
et al., Annals of Surgery 223(2):116-26 (1996); Dalesandro et al.,
J. Thorac. Cardi. Surg., 11(2):416-22 (1996); and Makarov et al.,
Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).
EXAMPLES
[0122] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
[0123] This example illustrates that RNA silencing is a natural
antiviral defense mechanism in animals and that certain animal
viruses encode RNA silencing suppressors to counter this defense
mechanism.
SUMMARY
[0124] RNA silencing is a sequence-specific RNA degradation
mechanism that is operational in plants and animals. Here we show
that flock house virus (FHV) is both an initiator and a target of
RNA silencing in Drosophila host cells and that FHV infection
requires suppression of RNA silencing by a FHV-encoded protein, B2.
These findings establish RNA silencing as a novel adaptive
antiviral defense in animal cells. B2 also inhibits RNA silencing
in transgenic plants, providing evidence for a conserved RNA
silencing pathway in the plant and animal kingdoms.
BACKGROUND
[0125] Posttranscriptional gene silencing, quelling and RNA
interference (RNAi) are mechanistically related RNA silencing
processes that destroy RNA in a sequence-specific manner (D.
Baulcombe, Curr. Biol., 12:R83 (2002); Hutvagner et al., Curr.
Opin. Genet. Dev., 12:225 (2002)).
[0126] Available data show that double-stranded (ds) RNA serves as
the initial trigger of RNA silencing and upon recognition, is
processed by the Dicer RNase into short fragments of 21 nucleotides
(nt) in length. These short interfering (si)RNAs are then
incorporated into a dsRNA-induced silencing complex (RISC) to guide
cycles of specific RNA degradation (D. Baulcombe, Curr. Biol.,
12:R83 (2002); Hutvagner et al., Curr. Opin. Genet. Dev., 12:225
(2002)). Here we report that RNA silencing plays a natural
antiviral role in animal cells as has been established in plants
(Vance et al., Science, 292:2277 (2001); Li et al., Curr. Opin.
Biotechnol., 12:150 (2001)).
[0127] The B2 gene of FHV shares key features, but not sequence
similarity, with the plant cucumoviral 2b gene (Ding et al., EMBO
J, 14:5762 (1995)), which encodes a known group of silencing
suppressors (Brigneti et al., EMBO J, 17:6739 (1998); Li et al.,
EMBO J, 18:2683 (1999)). Both open reading frame (ORF) 2b and B2
overlap the carboxyl terminal region and occupy the +1 reading
frame of the ORF encoding the viral RNA-dependent RNA polymerase,
and are translated in vivo via a subgenomic mRNA (Ding et al., EMBO
J, 14:5762 (1995)).
[0128] Methods
[0129] Full-length cDNA of FHV RNA1, together with a tobacco
ringspot virus satellite RNA ribozyme at the 3'-end (L. A. Ball, J
Virol, 69:720 (1995)), was cloned immediately downstream of the
CuSO.sub.4-inducible metallothionein promoter in pMT/V5-HisA vector
(Invitrogen) to give pRNA1 (see, FIG. 3). Precise fusion of the
RNA1 sequence at the +1 transcriptional site was achieved by
mutagenesis via PCR (Ding et al., EMBO J, 14:5762 (1995)).
[0130] pRNA1-AB2 was derived from pRNAl and contained the two point
mutations (T2739C and C2910A) described previously (L. A. Ball, J
Virol, 69:720 (1995)) that abolished the coding potential of ORF B2
but had no effect on the overlapping ORF A in the genomic RNA or
ORF B1 in RNA3. The full-length RNA3 sequence (see, FIG. 3) with
the start codon of the ORF B1 changed to AGC was cloned in
pMT/V5-HisA to yield pB2. The 3'-untranslated region of RNA3 up to
and including the stop codon in pB2 was replaced by a sequence
coding for the 6.times.His tag plus a stop codon at the 3'-end to
give pB2/His. Plasmid transfection and subsequent transcriptional
induction one day after transfection were carried out according to
manufacturer's recommendation.
[0131] RNAi procedure and dsRNAs corresponding to cyclinE, GFP,
AGO2, Dicer 1 and 2 were as described (Hammond et al., Science,
293:146 (2001); Bernstein et al., Nature, 411:494 (2001); Hammond
et al., Nature, 404:293 (2000)). dsRNA corresponding to the
3-terminal 500 nts of FHV RNA1 was generated similarly by in vitro
transcription.
[0132] Total RNAs were extracted from S2 cells two days after
induction and RNA blot probed with a cDNA corresponding to the
3'-terminal 1672 nts of RNA1 (FIG. 2D).
[0133] The AGO2 probe hybridized to the central region of AGO2 mRNA
that does not overlap the terminal regions targeted by dsRNAs.
[0134] FHV virion purification and infection of S2 cells were
carried out as described (Friesen et al., J. Virol., 42, 986
(1982)).
[0135] The riboprobe detecting the FHV-specific siRNAs was
complementary to the 3'-terminal 1672 nt of RNA1 (FIG. 2A). cDNA
probes used in FIG. 2B corresponded to nucleotides 2428-3002 of
RNA1 and nucleotides 469-1076 of RNA2. Reproducible results were
obtained from at least three independent infection/transfection
experiments.
[0136] Effect of B2 Deletion on GFP Protein Expression as Detected
by a Agrobacterium Co-Infiltration Assay
[0137] The FHV B2 protein exhibited a potent silencing suppression
activity (FIG. 1) in the Agrobacterium co-infiltration assay
(Voinnet et al., Cell, 103:157 (2000)), established in transgenic
plants expressing green fluorescent protein (GFP). Transient B2
expression prevented RNA silencing of the GFP transgene, leading to
a strong and prolonged green fluorescence examined under UV
illumination (FIG. 1, left), similar to suppression by the
cucumoviral 2b proteins (Guo et al., EMBO J, 21:398 (2002); FIG. 1,
right).
[0138] In contrast, a broad red fluorescent zone surrounding the
infiltrated patch (FIG. 1, middle) became clearly visible six days
post infiltration when the co-infiltrated transgene directed
translation of neither 2b nor B2.
[0139] Confirmation of Function of B2 in Plants by Northern
Blot
[0140] Overview
[0141] RNA blot hybridizations confirmed that expression of either
protein was associated with high accumulation levels of the GFP
mRNA. In addition, the GFP-specific siRNAs (Hamilton et al.,
Science, 286:950 (1999)) remained at extremely low levels in the
leaves where there was expression of either B2 or 2b.
[0142] Methods
[0143] cDNA to FHV RNA3 was cloned between the cauliflower mosaic
virus 35S promoter and terminator in a binary plasmid and
transformed into A. tuniefaciens as described (Guo et al., EMBO J,
21:398 (2002)). 35S-B2 and 35S-B1 directed expression of protein B2
and B1, respectively, as the initiation codon of ORFBI was changed
to AGC in 35S-B2 and that of ORFB2 was changed to ACG in 35S-B1.
35S-AB2 did not code for any FHV proteins as initiation codons of
both ORF B1 and B2 were changed to ATC. eDNAs encoding the tomato
aspermy virus 2b (T2b) and its untranslatable coding sequence
(TA2b) described previously (Li et al., EMBO J, 18:2683 (1999))
were similarly cloned to the binary vector to give 35S-T2b and
35S-T.DELTA.2b. A. tumefaciens strain 35S-GFP (Brigneti et al.,
EMBO J, 17:6739 (1998)) was mixed with either 35S-B2, 35S-B1,
35S-AB2, 35S-T2b, or 35S-T.DELTA.2b before infiltrated into the
leaves of the GFP-expressing N. benthamiana plants.
[0144] Results
[0145] FIG. 4A shows Northern blot analysis of total RNAs extracted
3 and 6 days after infiltration with 35S-GFP alone (-; lanes 1
& 6) or plus 35S-B2 (B2; lanes 2 & 7), 35S-B1 (B1; lanes 3
& 8), 35S-.DELTA.B2 (AB2; lanes 4 & 9), or 35S-T2b (T2b;
lanes 5 & 10). The filter was successively hybridized with
probes specific for the coding sequence of GFP (upper panel) and B2
(lower panel) as described (Guo et al., EMBO J, 21:398 (2002)).
Equivalent loading of the total plant RNA for each lane was
determined by methylene blue staining of the ribosomal RNA.
[0146] FIG. 4B shows the accumulation of GFP siRNAs. siRNAs were
extracted from the same samples described above and analyzed as
described (Guo et al., EMBO J, 21:398 (2002)). Lane 11 contained
three RNA species of 22, 23 and 26 nucleotides in length
transcribed in vitro from linearized plasmids by T7 RNA polymerase.
Note that when co-infiltrated with 35S-GFP, expression of either B2
(lanes 2 and 7) or T2b (lanes 5 and 10) was associated with high
accumulation levels of the GFP mRNA in infiltrated leaves (FIG. 2A,
upper panel). Further, the GFP-specific siRNAs (FIG. 2B)
accumulated to high levels in the 35S-GFP infiltrated leaves (lanes
1 and 6), but remained at extremely low levels in the leaves that
also expressed B2 (lanes 2 and 7) or T2b (lanes 5 and 10). In
contrast, when the co-infiltrated FHV-derived transgene directed
expression of no protein product (lanes 4 and 9) or the overlapping
B1 (lanes 3 and 8), the accumulation of siRNAs was as high as these
infiltrated with 35S-GFP alone. In addition, in vivo expression of
B2, but not of B1, was correlated with the accumulation of the
FHV-specific mRNA (FIG. 2A, lower panel) and the absence of the
FHV-specific siRNAs, indicating that mRNA derived from the
infiltrated FHV transgene was also targeted for silencing in the
infiltrated leaves.
[0147] Construction and Plant Infections of CMV Chimera
[0148] B2 was able to functionally substitute for 2b of cucumber
mosaic virus (CMV) in whole plant infections, as found previously
for a CMV 2b homologue (Ding et al., Proc Natl Acad Sci USA,
93:7470 (1996)).
[0149] Infectious CMV (Q strain) plasmid DNAs, pQCDl, 2 and 3 (Ding
et al., EMBO J, 14:5762 (1995)), were first cloned into a single
binary plasmid to give pBinCMV. pBinCMV-.DELTA.2b was obtained by
replacing the 2b coding sequence in pBinCMV with a unique SmaI
site. The coding sequence for B2 or T2b was then cloned to the
SniaI site to give pBinCMV-B2 and pBinCMV-2b. These binary plasmids
were transformed into A. tumefaciens and seedlings of N. tabacum
(cv. Samsun) were inoculated by Agrobacterium infiltration. Four, 7
and 14 days after inoculation, total RNAs were extracted from the
inoculated and systemically infected leaves and analyzed by
Northern hybridizations using a .sup.32P-labeled cDNA probe
corresponding to the 3' terminal 200 nucleotides of CMV RNA 2.
[0150] The results show that the defect of virus RNA accumulation
in the inoculated and systemically infected leaves, known to be
associated with the deletion of 2b (Ding et al., EMBO J, 14:5762
(1995); Ding et al., Proc Natl Acad Sci USA, 93:7470 (1996)), was
largely rescued by the expression of either B2 or T2b.
[0151] FHV Triggers RNA Silencing in Drosophila Cells
[0152] The finding that an FHV-encoded protein suppresses RNA
silencing in plants indicates that it also plays a role for RNA
silencing in FHV infections of animal hosts. FHV belongs to the
Nodaviridae family, members of which naturally infect vertebrate
and invertebrate hosts and Drosophila cells support complete
infection cycles of FHV (Ball et al., in Virus taxonomy--Seventh
report of the international committee on taxonomy of viruses. H. V.
van Regenmortel et al., Eds. (Academic Press, 2000), pp.747).
[0153] FIG. 2 shows that infection of Drosophila S2 cells with FHV
virions results in a rapid appearance of FHV-specific siRNAs of
both plus (FIG. 2A) and minus polarities. Accumulation of siRNA
trailed that of FHV genomic and subgenomic RNAs (FIG. 2C),
suggesting that the decreased accumulation of FHV RNAs at later
stages of FHV infection (Johnson et al., J Virol, 73:7933 (1999);
FIG. 2B) is due to FHV-specific RNA silencing.
[0154] ARGO2, Part of the RISC Complex, is a Component of the RNA
Silencing Pathway in Animals
[0155] To investigate this possibility, we constructed a
full-length FHV RNA1 cDNA clone, pRNA1 (see, FIG. 3), which after
transfection into S2 cells directed RNA1 self-replication and
transcription of RNA3 (L. A. Ball, J Virol, 69:720 (1995)), the
subgenomic mRNA for B2 (FIG. 2D, lane 2). We found that depleting
the mRNA of Argonaute2 (AGO2) by RNAi, an essential component of
the RISC complex (Hammond et al., Science, 293:146 (2001)), led to
a pronounced increase (2-3 fold) in the accumulation of FHV RNAs 1
and 3 (lanes 6-8) whereas co-transfection of cyclinE or GFP dsRNAs
with pRNA 1 had minimal effect (lanes 4-5), indicating that a
functional RNA silencing pathway naturally restricted FHV
accumulation in the host cells.
[0156] Furthermore, co-transfection of pRNA1 with a dsRNA targeting
the 3'-terminal 500 nucleotides of FHV RNA1 completely prevented
accumulation of intact FHV RNA1 in S2 cells (lane 3). These results
collectively demonstrate that FHV is both an initiator and target
of RNA silencing in this animal host.
[0157] B2 is Essential for FHV Accumulation in Drosophila Cells
[0158] Further studies showed that B2 was essential for FHV
accumulation in the Drosophila cells, which is in contrast to a
previous study carried out in non-host mammalian cells (L. A. Ball,
J Virol, 69:720 (1995)). A B2-knockout mutant of FHV RNAI, referred
to as RNA1-AB2, which contains the previously described point
mutations (L. A. Ball, J Virol, 69:720 (1995)) that converted the
first and 58th codons of the B2 ORF into Ser and stop codons
respectively, failed to accumulate to detectable levels after
transfection into S2 cells (lanes 12 and 20).
[0159] This defect was partially trans-complemented (up to 10% of
the wild type level) by co-transfection of a plasmid expressing
either B2 (lanes 13 and 21) or a His-tagged B2 (lane 22).
Expression of the His-tagged B2 from the co-transfected plasmid was
detected in S2 cells by Western blot analysis using an antibody
recognizing the His tag.
[0160] RT-PCR and sequencing revealed that the introduced mutations
were stably maintained in the progeny FHV RNAs extracted from
infected cells, indicating that B2 was indeed expressed from the
co-transfected plasmid rather than from a revertant RNA1.
[0161] B2 Suppresses FHV RNA Silencing in Drosophila Cells
[0162] Significantly, accumulation of RNA1-AB2 in S2 cells was
efficiently rescued, up to 50% of the wild type level, by
co-transfection with the AGO2 dsRNAs either singly (lanes 14-15) or
in combination (lane 16). However, co-transfection with dsRNAs
targeting mRNAs of the two Drosophila Dicer genes (Bernstein et
al., Nature, 411:494 (2001)), was not effective (lane 17) under the
same conditions. This is possibly due to a more efficient mRNA
depletion by RNAi for AGO2 (lanes 14-16) than Dicer (Hammond et
al., Science, 293:146 (2001); Bernstein et al., Nature, 411:494
(2001)), which is required to process the input dsRNA. Notably, the
level of complementation by RNAi of AGO2 was higher than that
achieved by the B2-expressing plasmid (lane 13), although still
less efficiently than B2 expression from wild type RNA1 (lane 10).
Therefore, in the absence of B2 expression, FHV RNAs 1 and 3
accumulated to substantial levels when the RISC complex was
disrupted by AGO2 depletion.
[0163] These data confirmed the previous finding (L. A. Ball, J
Virol, 69:720 (1995)) that B2 is not required for RNA1
self-replication and indicate that the essential function of B2 for
FHV infection of the S2 host cells observed in this study was to
suppress RNA silencing that targeted FHV RNAs for degradation.
CONCLUSIONS
[0164] Thus, the same protein blocks RNA silencing in both animals
and plants, providing the first experimental evidence for a highly
conserved RNA silencing pathway in different kingdoms. Notably,
FHV-B2 did not prevent initiation of virus RNA silencing (FIG. 2A)
or RNAi (FIG. 2D), and thus may be functionally analogous to the
plant viral 2b protein (Guo et al., EMBO J, 21:398 (2002)).
[0165] It is known that RNA interference operates in animals
including mammals (D. Baulcombe, Curr. Biol., 12:R83 (2002);
Hutvagner et al., Curr. Opin. Genet. Dev., 12:225 (2002); Elbashir
et al., Nature, 411:494 (2001)). In this work, we demonstrate that
infection of Drosophila cells with an RNA virus triggers strong
virus RNA silencing and that the same virus is equipped with an
effective silencing suppressor essential for infection. These data
provide compelling, direct evidence that RNA silencing naturally
acts as a novel adaptive antiviral defense in animal cells. The
specificity mechanism of this adaptive defense is based on nucleic
acid base pairing between siRNA and its target RNA (D. Baulcombe,
Curr. Biol., 12:R83 (2002); Hutvagner et al., Curr. Opin. Genet.
Dev., 12:225 (2002)), and thus is distinct from cellular and
humoral adaptive immunity based on peptide recognition (J. L.
Whitton, M. B. A. Oldstone, in Fields Virology, D. M. Knipe, P. M.
Howley, Eds. (Lippincott Williams & Wilkins, 2001), vol. 1,
chap. 11. [fourth edition]).
[0166] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
2 1 160 PRT Hepatitis C virus F protein 1 Ala Arg Ile Leu Asn Leu
Lys Lys Lys Thr Asn Val Thr Pro Thr Val 1 5 10 15 Ala His Arg Thr
Ser Ser Ser Arg Val Ala Val Arg Ser Leu Val Glu 20 25 30 Phe Thr
Cys Cys Arg Ala Gly Ala Leu Asp Trp Val Cys Ala Arg Arg 35 40 45
Glu Arg Leu Pro Ser Gly Arg Asn Leu Glu Val Asp Val Ser Leu Ser 50
55 60 Pro Arg Leu Val Gly Pro Arg Ala Gly Pro Gly Leu Ser Pro Gly
Thr 65 70 75 80 Leu Gly Pro Ser Met Ala Met Arg Ala Ala Gly Gly Arg
Asp Gly Ser 85 90 95 Cys Leu Pro Val Ala Leu Gly Leu Ala Gly Ala
Pro Gln Thr Pro Gly 100 105 110 Val Gly Arg Ala Ile Trp Val Arg Ser
Ser Ile Pro Leu Arg Ala Ala 115 120 125 Ser Pro Thr Ser Trp Gly Thr
Tyr Arg Ser Ser Ala Pro Leu Leu Glu 130 135 140 Ala Leu Pro Gly Pro
Trp Arg Met Ala Ser Gly Phe Trp Lys Thr Ala 145 150 155 160 2 540
RNA Hepatitis C virus F protein 2 augagcacga auccuaaacc ucaaaaaaaa
aacaaacgua acaccaaccg ucgcccacag 60 gacgucaagu ucccgggugg
cggucagauc guugguggag uuuacuuguu gccgcgcagg 120 ggcccuagau
ugggugugcg cgcgacgaga aagacuuccg agcggucgca accucgaggu 180
agacgucagc cuauccccaa ggcucgucgg cccgagggca ggaccugggc ucagcccggg
240 uacccuuggc cccucuaugg caaugagggc ugcggguggg cgggauggcu
ccugucuccc 300 cguggcucuc ggccuagcug gggccccaca gacccccggc
guaggucgcg caauuugggu 360 aaggucaucg auacccuuac gugcggcuuc
gccgaccuca ugggguacau accgcucguc 420 ggcgccccuc uuggaggcgc
ugccagggcc cuggcgcaug gcguccgggu ucuggaagac 480 ggcgugaacu
augcaacagg gaaccuuccu gguugcucuu ucucuaucuu ccuucuggcc 540
* * * * *