U.S. patent application number 10/127318 was filed with the patent office on 2002-08-29 for methods of screening for antiviral compounds.
This patent application is currently assigned to UAB Research Foundation. Invention is credited to Hardy, Richard W., Wertz, Gail W..
Application Number | 20020119446 10/127318 |
Document ID | / |
Family ID | 22401601 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119446 |
Kind Code |
A1 |
Wertz, Gail W. ; et
al. |
August 29, 2002 |
Methods of screening for antiviral compounds
Abstract
The present invention demonstrates that the M2-1 protein of
respiratory syncytial virus has a conserved Cys.sub.3--His.sub.1
motif known to bind zinc ions in other proteins and that mutations
of the predicted zinc coordinating residues in the
Cys.sub.3--His.sub.1 motif affect the transcriptional
antitermination activity of M2-1, its ability to interact with
nucleocapsid protein, and the phosphorylation state of M2-1. This
invention clearly demonstrates the requirement for conservation of
the Cys.sub.3--His.sub.1 motif in order to maintain the functional
integrity of the M2-1 protein. Therefore, the present invention
provides for methods of designing and screening compounds for
antiviral activity towards respiratory syncytial virus based upon
the loss of function of the M2-1 protein.
Inventors: |
Wertz, Gail W.; (Birmingham,
AL) ; Hardy, Richard W.; (Maplewood, MO) |
Correspondence
Address: |
Benjamin Aaron Adler
ADLER & ASSOCIATES
8011 Candle Lane
Houston
TX
77071
US
|
Assignee: |
UAB Research Foundation
|
Family ID: |
22401601 |
Appl. No.: |
10/127318 |
Filed: |
April 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10127318 |
Apr 22, 2002 |
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09511023 |
Feb 23, 2000 |
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6376171 |
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60122251 |
Feb 26, 1999 |
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Current U.S.
Class: |
435/5 ; 530/350;
530/826; 536/23.72 |
Current CPC
Class: |
G01N 2500/00 20130101;
G01N 2333/135 20130101; C12N 2760/18522 20130101; C07K 14/005
20130101; G01N 33/56983 20130101 |
Class at
Publication: |
435/5 ; 435/6;
530/350; 530/826; 536/23.72 |
International
Class: |
C12Q 001/70; C12Q
001/68; C07H 021/04; C07K 001/00; C07K 014/00; C07K 017/00 |
Goverment Interests
[0002] This invention was produced in part using funds obtained
through grants AI12464 and AI20181 from the National Institute of
Health. Consequently, the federal government has certain rights in
this invention.
Claims
What is claimed is:
1. A method of screening for antiviral compounds directed towards
respiratory syncytial virus, comprising the steps of: a) treating a
sample of respiratory syncytial virus with a compound, thereby
producing a treated sample and a n untreated sample; b) producing
respiratory syncytial virus RNA transcripts in the presence of said
treated sample or in the presence of said untreated sample; and c)
comparing said transcripts produced in the presence of said treated
sample with said transcripts produced in the presence of said
untreated sample, wherein less readthrough transcripts due to
termination at gene end signal produced in the presence of said
treated sample is indicative of an antiviral compound directed
towards respiratory syncytial virus.
2. The method of claim 1, wherein said sample of respiratory
syncytial virus is selected from the group consisting of a purified
respiratory syncytial virus M2-1 protein, M2-1 protein produced in
cell from an expression vector, an isolated respiratory syncytial
virus, a respiratory syncytial virus-infected cell and a
respiratory syncytial virus-infected animal.
3. A method of screening for antiviral compounds directed towards
respiratory syncytial virus, comprising the steps of: a) treating a
sample of respiratory syncytial virus with a compound, thereby
producing a treated sample and an untreated sample; and b)
comparing said treated sample with said untreated sample, wherein
an inhibitory effect on said treated sample of a characteristic
selected from the group consisting of transcriptional
antitermination by M2-1 protein, zinc binding of M2-1 protein,
phosphorylation of M2-1 protein, M2-1 protein binding to
respiratory syncytial virus N protein, viral transcription, viral
replication and generation of progeny virus particles is indicative
of an antiviral compound directed towards respiratory syncytial
virus.
4. The method of claim 3, wherein said sample of respiratory
syncytial virus is selected from the group consisting of a purified
respiratory syncytial virus M2-1 protein, M2-1 protein produced in
cell from an expression vector, an isolated respiratory syncytial
virus, a respiratory syncytial virus-infected cell and a
respiratory syncytial virus-infected animal.
5. A method of screening for antiviral compounds directed towards
respiratory syncytial virus, comprising the steps of: a) treating a
sample of respiratory syncytial virus with a chelator or a compound
that inhibits zinc binding, thereby producing a treated sample and
an untreated sample; and b) comparing said treated sample with said
untreated sample, wherein an inhibitory effect on said treated
sample of a characteristic selected from the group consisting of
transcriptional antitermination by M2-1 protein, zinc binding of
M2-1 protein, phosphorylation of M2-1 protein, M2-1 protein binding
to respiratory syncytial virus N protein, viral transcription,
viral replication and generation of progeny virus particles is
indicative of an antiviral compound directed towards respiratory
syncytial virus.
6. The method of claim 5, wherein said inhibition of zinc binding
is selected from the group consisting of competing with zinc for
binding to the Cys.sub.3--His.sub.1 motif within the M2-1 protein,
preventing zinc from binding, destroying the formation of the
Cys.sub.3--His.sub.1 motif, and interfering with the interaction of
the properly formed Cys.sub.3--His.sub.1 motif with its target.
7. The method of claim 5, wherein said sample of respiratory
syncytial virus is selected from the group consisting of a purified
respiratory syncytial virus M2-1 protein, M2-1 protein produced in
cell from an expression vector, an isolated respiratory syncytial
virus, a respiratory syncytial virus-infected cell and a
respiratory syncytial virus-infected animal.
8. A method of screening for antiviral compounds directed towards
respiratory syncytial virus, comprising the steps of: a) treating a
sample of respiratory syncytial virus with a compound, thereby
producing a treated sample and a n untreated sample; and b)
producing respiratory syncytial virus RNA transcripts in the
presence of said treated sample or in the presence of said
untreated sample, wherein an inhibition of virus RNA transcription
or production of progeny virus particles in the presence of said
treated sample is indicative of an antiviral compound directed
towards respiratory syncytial virus.
9. The method of claim 8, wherein said sample of respiratory
syncytial virus is selected from the group consisting of core
polymerase protein, nucleocapsid protein, phosphoprotein, an
isolated respiratory syncytial virus, a respiratory syncytial
virus-infected cell and a respiratory syncytial virus-infected
animal.
10. A method of designing antiviral compounds directed towards
respiratory syncytial virus, comprising the steps of: a) designing
a compound that inhibits zinc binding to a Cys.sub.3--His.sub.1
motif of respiratory syncytial virus M2-1 protein; b) treating a
sample of respiratory syncytial virus with said compound, thereby
producing a treated sample and an untreated sample; and c)
comparing said treated sample with said untreated sample, wherein
an inhibitory effect on said treated sample of a characteristic
selected from the group consisting of transcriptional
antitermination by M2-1 protein, zinc binding of M2-1 protein,
phosphorylation of M2-1 protein, M2-1 protein binding to
respiratory syncytial virus N protein, viral transcription, viral
replication and generation of progeny virus particles is indicative
of an antiviral compound directed towards respiratory syncytial
virus.
11. The method of claim 10, wherein said inhibition of zinc binding
is selected from the group consisting of competing with zinc for
binding to the Cys.sub.3--His.sub.1 motif, preventing zinc from
binding, destroying the formation of the Cys.sub.3--His.sub.1
motif, and interfering with the interaction of the properly formed
Cys.sub.3--His.sub.1 motif with its interacting target.
12. The method of claim 10, wherein said sample of respiratory
syncytial virus is selected from the group consisting of a purified
respiratory syncytial virus M2-1 protein, M2-1 protein produced in
cell from an expression vector, an isolated respiratory syncytial
virus, a respiratory syncytial virus-infected cell and a
respiratory syncytial virus-infected animal.
13. The method of claim 10, wherein said designing is done by
computer modeling.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims benefit of provisional patent
application U.S. Ser. No. 60/122,251, filed Feb. 26, 1999.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the field of
virology and to the process and control of viral RNA transcriptin
More specifically, the present invention relates to a
Cys.sub.3--His.sub.1 motif within the M2-1 protein of respiratory
syncytial virus and the use of this functional motif as a target
for screening of antiviral agents.
[0005] 2. Description of the Related Art
[0006] Human respiratory syncytial (RS) virus is a member of the
pneumovirus genus of the Paramyxoviridae. It is the leading viral
cause of pediatric lower respiratory tract disease and is a
significant cause of morbidity and mortality worldwide. The genome
of RS virus is a single strand of negative-sense RNA 15,222
nucleotides in length having 10 genes encoding 11 proteins (4, 15,
25, 33). As with all nonsegmented, negative-sense RNA viruses, RNA
synthesis requires a genomic RNA encapsidated with nucleocapsid (N)
protein and the virus encoded components of the RNA dependent RNA
polymerase, the phosphoprotein (P) and the large polymerase protein
(L) (7, 38). The N, P and L proteins are sufficient for replication
of the genomic RNA (10, 38). However, RS virus, unlike other
nonsegmented negative-sense RNA viruses, encodes an additional
protein, M2-1, which functions during transcription of the viral
mRNAs (3). This protein has been shown to increase the processivity
of the viral polymerase thus preventing premature termination
during transcription. Additionally it has been shown that the M2-1
protein enhances readthrough of transcription termination signals
and thus functions as a transcription antiterminator (8, 13).
Furthermore, the RS virus gene end sequences vary and it has been
shown that the M2-1 protein acts differentially at the different
gene ends ( 11)
[0007] The M2-1 protein is found only in pneumoviruses. It is
encoded by the first of two open reading frames (ORF) in the next
to last gene of the RS virus genome and is 194 amino acids in
length (calculated m.w.=22,150 daltons) (5). M2-1 is a hydrophilic
protein with a predicted p1 of 9.6. Examination of the predicted
amino acid sequence led to the identification of a
Cys.sub.3--His.sub.1 motif (C--X.sub.7--C--X.sub.5--C-
--X.sub.3--H) located near the amino terminus of the protein from
residues 7 to 25. This motif is found in the M2-1 protein of all t
h e pneumoviruses examined to date (12, 20, 37, 39). A similar
motif is found in VP30 of the filoviruses (29). A number of motifs
have been characterized as coordinating the binding of zinc. These
motifs have been grouped according to the arrangement and number of
cysteine and histidine residues involved in coordinating the zinc
ion. Many proteins bind zinc, and, in a number of enzymes, zinc has
been shown to play a role in catalysis (16). However, in other
cases, zinc plays a purely structural role (24). The
Cys.sub.3--His.sub.1 motif has been characterized in only one
protein, Nup475, a mammalian transcription factor in which it was
demonstrated to bind zinc and a structure for this motif has been
proposed using NMR an d photometric analyses (36).
[0008] Six species of the RS virus M2-1 protein have been observed
by two-dimensional electrophoresis (27). When the M2-1 protein was
analyzed by SDS-PAGE under reducing conditions, the majority of the
protein was found in two forms distinguished b y their
electrophoretic mobility. The cause of the differences in migration
of these species or whether the different species have different
functions is unknown. The M2-1 protein has been reported to be
phosphorylated, but the relationship between phosphorylation and
the different forms of the protein has not been investigated
(18).
[0009] The M2-1 protein has also been shown to interact with the N
protein in RS virus-infected cells or when the two proteins are
coexpressed in cells from plasmid vectors (9, 28). The significance
of this interaction and the role it may play is currently
unknown.
[0010] The prior art is deficient in methods of screening for
antiviral compounds that are specific for respiratory syncytial
virus. The present invention fulfills this long-standing need and
desire in the art.
SUMMARY OF THE INVENTION
[0011] In the instant invention the role of the predicted zinc
coordinating residues of the Cys.sub.3--His.sub.1 motif in the
antitermination activity of M2-1 protein, in its ability to
interact with N protein, and in its phosphorylation state were
examined. It was found that mutations of the residues predicted to
coordinate zinc prevented the M2-1 protein from enhancing
transcriptional readthrough and interacting with the nucleocapsid
protein. It was also found that the two major species of the M2-1
protein distinguished by their mobility in reducing SDS-PAGE
differed according to whether they were phosphorylated. This work
demonstrates the requirement for conservation of the
Cys.sub.3--His.sub.1 motif, a potential zinc binding domain, to
maintain the functional integrity of the M2-1 protein.
[0012] One object of the present invention is to provide a method
of screening for antiviral compounds specific for respiratory
syncytial virus.
[0013] In one embodiment of the present invention, there is
provided a method of screening for antiviral compounds directed
towards respiratory syncytial virus, comprising the steps of: a)
treating a sample of respiratory syncytial virus with a compound,
thereby producing a treated sample and an untreated sample; b)
producing respiratory syncytial virus RNA transcripts in the
presence of said treated sample or in the presence of said
untreated sample; and c) comparing said transcripts produced in the
presence of said treated sample with said transcripts produced in
the presence of said untreated sample, wherein less readthrough
transcripts due to termination at gene end signal produced in the
presence of said treated sample is indicative of an antiviral
compound directed towards respiratory syncytial virus.
[0014] In another embodiment of the present invention, there is
provided a method of screening for antiviral compounds directed
towards respiratory syncytial virus, comprising the steps of: a)
treating a sample of respiratory syncytial virus with a compound,
thereby producing a treated sample and an untreated sample; and b)
comparing the treated sample with an untreated sample, wherein a n
inhibitory effect on the treated sample compared to the untreated
sample of a characteristic such as M2-1 transcriptional
antitermination, zinc binding, phosphorylation, binding to
respiratory syncytial virus N protein, viral transcription or
generation of progeny virus particles is indicative of a compound
with antiviral activity.
[0015] In another embodiment of the present invention, there is
provided a method of screening for antiviral compounds directed
towards respiratory syncytial virus, comprising the steps of: a)
treating a sample of respiratory syncytial virus with a chelator or
a compound that inhibits binding of Zinc; and b) comparing the
treated sample with an untreated sample, wherein an inhibitory
effect on the treated sample compared to the untreated sample of a
characteristic such as M2-1 transcriptional antitermination, zinc
binding, phosphorylation, binding to respiratory syncytial virus N
protein, viral transcription or generation of progeny virus
particles is indicative of a compound with antiviral activity.
[0016] In yet another embodiment of the present invention, there is
provided a method of screening antiviral compounds directed towards
respiratory syncytial virus, comprising the steps of: a) treating a
sample of respiratory syncytial virus selected from the group
consisting of core polymerase protein, nucleocapsid protein,
phosphoprotein, an isolated virus or a virus-infected cell with a
compound, thereby producing a treated sample and an untreated
sample; and b) producing respiratory syncytial virus RNA
transcripts in the presence of said treated sample or in the
presence of said untreated sample, wherein an inhibition of virus
RNA transcription or production of progeny virus particles in the
presence of said treated sample is indicative of an antiviral
compound directed towards respiratory syncytial virus.
[0017] In yet another embodiment of the present invention, there is
provided a method of designing antiviral compounds directed towards
respiratory syncytial virus, comprising the steps of: a) designing
a compound that inhibits zinc binding to a Cys.sub.3--His.sub.1
motif of a respiratory syncytial virus M2-1 protein; b) treating a
sample of respiratory syncytial virus with said designed compound;
and c) comparing said treated sample with said untreated sample,
wherein an inhibitory effect on said treated sample, when compared
to the untreated sample, of a characteristic such as M2-1
transcriptional antitermination, zinc binding, phosphorylation,
binding to respiratory syncytial virus N protein, viral
transcription or generation of progeny virus particles is
indicative of a compound with antiviral activity. A preferred
method of designing is b y computer modeling.
[0018] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention. These
embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions of the invention briefly summarized
above may be had by reference to certain embodiments thereof which
are illustrated in the appended drawings. 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 therefore should not be considered to limit the scope of the
invention.
[0020] FIG. 1 shows the effects of mutations in the
Cys.sub.3--His.sub.1 motif of the M2-1 protein on RS virus
transcription.
[0021] FIG. 1A shows a diagram of the mutations made in the
Cys.sub.3--His.sub.1 motif. NH.sub.2 signifies the amino terminus
of the M2-1 protein. The predicted zinc coordinating residues are
identified by an asterisk.
[0022] FIG. 1B shows a diagram of the RS virus dicistronic
subgenomic replicon containing the M/SH intergenic junction used in
the transcription assay and the potential products of
transcription.
[0023] FIG. 1C shows the products of RNA synthesis from the M/SH
subgenomic replicon in the presence of wild-type (wt) or mutated
M2-1 proteins. Cells infected with recombinant MVA vaccinia virus
expressing T7 RNA polymerase, were transfected with pM/SH, pN, pP,
pL, and plasmids expressing wild type or mutant M2-1 proteins as
indicated. Cells were exposed to [.sup.3H]-uridine in the presence
of actinomycin-D and cytosine arabinoside. Total RNA was phenol
extracted, ethanol precipitated and analyzed by agarose-urea gel
electrophoresis followed by fluorography. rep, replication
products; r/t, products of readthrough transcription.
[0024] FIG. 2 shows the expression of M2-1 proteins with mutations
in the Cys.sub.3--His.sub.1 motif. HEp-2 cells were infected with
vTF7-3 and then transfected with plasmids encoding the wild type or
mutant M2-1 proteins. Cells were labeled with [.sup.35S]-methionine
and cysteine for 2 h at 16 h post-transfection. Cytoplasmic
extracts were prepared and proteins were immunoprecipitated with
the M2-specific MAb, 5H5. Labeled proteins from mock-transfected
cells (lane 2) and cells transfected with plasmids encoding the
M2-1 proteins (lanes 3 to 11) were analyzed by SDS-PAGE in 11 %
polyacrylamide gels, followed by fluorography. Lane 1,
[.sup.14C]-1abeled molecular size markers; molecular sizes in
kilodaltons are shown on the left of the gel. Position of the M2-1
protein(s) is indicated. wt, wild type.
[0025] FIG. 3 shows a pulse-chase analysis of wild-type M2-1
protein expression. HEp-2 cells infected with vTF7-3 were
transfected with a plasmid expressing the wild-type M2-1 protein.
Cells were exposed to [.sup.35S]-methionine and cysteine for
15-min. at 16-h post-transfection. Following the 15-min. labeling
period, cells were either harvested (lanes 1 and 2), or the medium
was removed and medium containing unlabeled methionine and cysteine
was added for 15 min. (lane 3), 30 min. (lane 4), or 60 min. (lane
5) prior to harvest. Labeled proteins were immunoprecipitated from
cytoplasmic extracts using M2-1 specific MAb 1C13.
Immunoprecipitated proteins were analyzed by SDS-PAGE under
reducing conditions in 11% polyacrylamide gels followed b y
fluorography. vv, mock transfection.
[0026] FIG. 4 shows the effects of mutations in the
Cys.sub.3--His.sub.1 motif of the M2-1 on phosphorylation. HEp-2
cells infected with vTF7-3 were mock-transfected (mock; lanes 2 and
3) or transfected with plasmid encoding wild type or mutant M2-1
proteins as indicated (lanes 4 to 9), or HEp-2 cells infected with
RS virus (RSV; lanes 10 to 13). Cells were exposed to
[.sup.35S]-methionine and cysteine (S; lanes 3, 5, 7, 9, 11, and
13) or [.sup.33P]-inorganic phosphate (P; lanes 2, 4, 6, 8, 10, and
12) for 2h at 16h post-transfection or 20 h post-infection. Labeled
proteins were immunoprecipitated using M2-1 specific MAb 5H5 (lanes
2 to 11), or anti-RS virus polyclonal serum (aRSV; lanes 12 and 13)
and analyzed by SDS-PAGE in 1% polyacrylamide gels followed by
fluorography. Positions of RS virus proteins are shown (lane 13).
The exposure time of lanes 10 to 13 was two times that of lanes 1
to 9. m.w., molecular size markers, size in kilodaltons. wt, wild
type.
[0027] FIG. 5 shows the effects of mutations in the
Cys.sub.3--His.sub.1 motif of the M2-1 protein on interaction with
the N protein. HEp-2 cells infected with vTF7-3 were transfected
with pN alone (lane 1) or pN and plasmids encoding wild-type or
mutated M2-1 proteins as indicated (lanes 2 to 6). Cells were
exposed to [.sup.35S]-methionine and cysteine for 2 h at 16 h
post-transfection. Labeled proteins were immunoprecipitated from
cytoplasmic extracts using M2-1 specific MAb 5H5.
Immunoprecipitated proteins were analyzed by SDS-PAGE in 11%
polyacrylamide gels followed by fluorography. Positions of the M2-1
and N proteins are indicated. wt, wild-type.
[0028] FIG. 6 shows a diagram of plasmid pWT5 and the RNAs
predicted to be synthesized from the monocistronic subgenomic
replicon that it encodes. The subgenomic replicon transcribed from
pWT5 includes the RS virus 3' and 5' termini flanking a fused
partial NSl and L gene. Thus, transcription by T7 polymerase
followed by self-cleavage gave rise to a negative-sense copy of the
subgenomic replicon RNA. This RNA can act as a template from which
the RS virus RNA polymerase can synthesize a 547-nucleotide (nt)
mRNA (not including the poly(A) tail) or a positive-sense product
of replication. tr, trailer.
[0029] FIG. 7 shows the products of RNA synthesis from WT5
subgenomic replicon. Cells were infected with vTF7-3, transfected
with cDNAs pWT5, pN, pP, pL and pM2-1 as indicated, and exposed to
[.sup.3H]uridine in the presence of actinomycin D. "O" indicated
that a particular plasmid has been omitted.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The M2 gene of respiratory syncytial (RS) virus has two open
reading frames (ORFs). ORFI encodes a 22 kDa protein termed M2-1.
The M2-1 protein contains a CyS.sub.3--His.sub.1 motif
(C--X.sub.7--C--X.sub.5--C--X.sub.3--H) near the amino terminus.
This motif is conserved in all strains of human, bovine and ovine
species of RS virus. A similar motif found in the mammalian
transcription factor Nup475 has been shown to bind zinc. The M2-1
protein of human RS virus functions as a transcription factor which
increases polymerase processivity and it enhances readthrough of
intergenic junctions during RS virus transcription thereby acting
as a transcription antiterminator. The M2-1 protein also interacts
with the nucleocapsid protein. The effects of mutations of cysteine
and histidine residues predicted to coordinate zinc in the
Cys.sub.3--His.sub.1 motif of M2-1 protein on transcription
antitermination and N protein binding were examined. It was found
that mutating the predicted zinc coordinating residues, the
cysteine residues at amino acid positions 7 and 15 or the histidine
residue at position 25, prevented M2-1 from enhancing
transcriptional readthrough. In contrast, mutations of amino acids
within this motif not predicted to coordinate zinc had no effect.
Mutations of the predicted zinc coordinating residues in the
Cys.sub.3--His.sub.1 motif also prevented M2-1 from interacting
with the nucleocapsid protein. One mutation of a non-coordinating
residue in the motif which did not affect readthrough during
transcription, E10G, prevented interaction with the nucleocapsid
protein. This suggests that M2-1 does not require interaction with
the nucleocapsid protein in order to function during transcription.
Analysis of the M2-1 protein in reducing SDS-PAGE revealed two
major forms distinguished by their mobility. The slower migrating
form was shown to be phosphorylated, whereas the faster migrating
form was not. Mutations in the Cys.sub.3--His.sub.1 motif caused a
change in distribution of the M2-1 protein from the slower to the
faster migrating form. The data presented here show that the
Cys.sub.3--His.sub.1 motif of M2-1 is essential for maintaining the
functional integrity of the protein and are consistent with this
motif coordinating the binding of zinc.
[0031] The present invention is drawn to a method of screening for
antiviral compounds directed towards respiratory syncytial virus,
comprising the steps of: a) treating a sample of respiratory
syncytial virus with a compound, thereby producing a treated sample
and an untreated sample; b) producing respiratory syncytial virus
RNA transcripts in the presence of said treated sample or in the
presence of said untreated sample; and c) comparing said
transcripts produced in the presence of said treated sample with
said transcripts produced in the presence of said untreated sample,
wherein less readthrough transcripts due to termination at gene end
signal produced in the presence of said treated sample is
indicative of an antiviral compound directed towards respiratory
syncytial virus. Representative samples of respiratory syncytial
virus include a purified M2-1 protein, a M2-1 protein produced in
cells from a vector, an isolated respiratory syncytial virus, a
respiratory syncytial virus-infected cell or a respiratory
syncytial virus-infected animal.
[0032] The present invention is also directed towards a method of
screening for antiviral compounds directed towards respiratory
syncytial virus, comprising the steps of: a) treating a sample of
respiratory syncytial virus with a compound, thereby producing a
treated sample and an untreated sample; and b) comparing the
treated sample with an untreated sample, wherein an inhibitory
effect on the treated sample compared to the untreated sample of a
characteristic such as M2-1 transcriptional antitermination, zinc
binding, phosphorylation, binding to respiratory syncytial virus N
protein, viral transcription or generation of progeny virus
particles is indicative of a compound with antiviral activity.
[0033] The present invention is also directed towards a method of
screening for antiviral compounds directed towards respiratory
syncytial virus, comprising the steps of: a) treating a sample of
respiratory syncytial virus with a chelator or a compound that
inhibits binding of Zinc; and b) comparing the treated sample with
a n untreated sample, wherein an inhibitory effect on the treated
sample compared to the untreated sample of a characteristic such as
M2-1 transcriptional antitermination, zinc binding,
phosphorylation, binding to respiratory syncytial virus N protein,
viral transcription or generation of progeny virus particles is
indicative of a compound with antiviral activity. Preferably, the
inhibition of zinc binding is the result of competing with zinc for
binding to the Cys3--His.sub.1 motif, preventing zinc from binding,
destroying the formation of the Cys3--His1 motif, or interfering
with the interaction of the properly formed Cys3--His1 motif with
its interacting target.
[0034] The present invention is also directed towards a method of
screening for antiviral compounds directed towards respiratory
syncytial virus, comprising the steps of: a) treating a sample of
respiratory syncytial virus selected from the group consisting of
core polymerase protein, nucleocapsid protein, phosphoprotein, a n
isolated virus or a virus-infected cell with a compound, thereby
producing a treated sample and an untreated sample; and b)
producing respiratory syncytial virus RNA transcripts in the
presence of said treated sample or in the presence of said
untreated sample, wherein an inhibition of virus RNA transcription
or production of progeny virus particles in the presence of said
treated sample is indicative of an antiviral compound directed
towards respiratory syncytial virus.
[0035] The present invention is further directed towards a method
of designing antiviral compounds directed towards respiratory
syncytial virus, comprising the steps of: a) designing a compound
that inhibits zinc binding to a Cys.sub.3--His.sub.1 motif of a
respiratory syncytial virus M2-1 protein; b) treating a sample of
respiratory syncytial virus with said designed compound; and c)
comparing said treated sample with said untreated sample, wherein
an inhibitory effect on said treated sample, when compared to the
untreated sample, of a characteristic such as M2-1 transcriptional
antitermination, zinc binding, phosphorylation, binding to
respiratory syncytial virus N protein, viral transcription or
generation of progeny virus particles is indicative of a compound
with antiviral activity. Preferably, the inhibition of zinc binding
is the result of competing with zinc for binding to the
Cys.sub.3--His.sub.1 motif, preventing zinc from binding,
destroying the formation of the Cys.sub.3--His.sub.1 motif, or
interfering with the interaction of the properly formed
Cys.sub.3--His.sub.1 motif with its interacting target. A preferred
method of designing is by computer modeling.
[0036] As used herein, the term "antiviral compound" refers to any
substance which inhibits the replication of a virus or inhibits an
y essential process in the replication cycle.
[0037] As used herein, the term "transcriptional antitermination"
refers to the function of the M2-1 protein as a transcription
factor which increases polymerase processivity and it enhances
readthrough of intergenic junctions during RS virus transcription
thereby acting as a transcription antiterminator.
[0038] As used herein, the term "zinc binding" refers to the
tetrahedral coordination of a zinc ion within a protein by the `R`
groups of cysteine and histidine residues.
[0039] As used herein, the term "phosphorylation" refers to the
addition of at least one phosphate group to the M2-1 protein via a
covalent bond.
[0040] As used herein, "binding to respiratory syncytial virus N
protein" refers to the interaction of the M2-1 protein with the
respiratory syncytial virus N protein to generate a stable
association of the N protein with the M2-1 protein (i.e. a complex
of M2-1 and N).
[0041] 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:
EXAMPLE 1
[0042] cDNA Constructs
[0043] Vectors for expressing the M2-1 protein in eukaryotic cells
were generated using a vaccinia T7 expression system. Generation of
cDNAs expressing N, P, L, and wild-type M2-1 proteins (pN, pP, pL,
and pORFI, respectively) has been described previously (13, 38).
Mutations in the Cys.sub.3--His.sub.1 motif of the M2-1 protein
were generated by PCR mutagenesis utilizing primers containing
coding changes, and cloned into the BamnHI/HindIII sites of pGEM3
behind the T7 promoter. Nucleotide sequences of cDNA constructs
were determined by dideoxy nucleotide chain termination DNA
sequencing. The generation of pM/SH (encoding an RS virus
dicistronic subgenomic replicon) was described previously (13).
EXAMPLE 2
[0044] Virus and Cells
[0045] HEp-2 cells were grown in minimum essential medium (MEM)
(GIBCO laboratories) supplemented with 5% heat-inactivated fetal
bovine serum (FBS) in 60-mm dishes. The A-2 strain of human RS
virus was propagated in HEp-2 cells. RS virus was added to the
cells at a m.o.i. of 1 p.f.u./cell. Virus was allowed to adsorb for
2 h a t 37.degree. C. Fresh medium (2 mls of MEM supplemented with
5% FBS) was added, and cells were incubated at 37.degree. C. for 18
h. The medium was removed, and medium deficient in methionine and
cysteine or phosphate as required was added for 30 min. Proteins
were labeled for 2 h using 66 uCi/ml [.sup.35S]-methionine and
cysteine (Tran.sup.35S-1abel, ICN) or 100 uCi/ml
[.sup.33P]-inorganic phosphate (ICN). Cells were harvested, and
cytoplasmic extracts were prepared as previously described (35). RS
virus specific proteins were detected b y immunoprecipitation
followed by SDS-PAGE.
EXAMPLE 3
[0046] cDNA Transfections
[0047] RS virus RNA synthesis, programmed by subgenomic replicons,
was assayed using a recombinant vaccinia virus T7 expression
system. HEp-2 cells infected with recombinant MVA vaccinia virus
expressing T7 RNA polymerase were transfected with 6 ug of pM/SH, 5
ug of pN, 2 ug of pP, 2 ug of pL, and 0.3 ug pORFI (encoding
wild-type M2-1 protein) or pGEM-based plasmid encoding mutant M2
protein. RS virus-specific RNAs were labeled with [.sup.3H] uridine
(33 uCi/ml; Moravek) in the presence of actinomycin D (10 ug/ml;
Sigma) and cytosine arabinoside (50 ug/ml; Sigma) at 16 h
post-transfection. After a 5-h labeling period, cells were
harvested and cytoplasmic extracts were prepared as previously
described (26). RNAs were purified by phenol extraction followed by
ethanol precipitation. RNAs were analyzed by electrophoresis in
1.75% agarose-urea gels and detected by fluorography (19, 34).
[0048] Expression of M2-1 mutant proteins was analyzed b y
transfecting vTF7-3-infected HEp-2 cells with 2 ug of pGEM-based
plasmids encoding the wild-type M2-1 protein or mutant M2-1
proteins. The interaction of the M2-1 protein with the nucleocapsid
protein was analyzed in cells cotransfected with the M2-1 plasmids
encoding wild-type or mutant M2-1 proteins and 5 ug pN.
Sixteen-hours post-transfection cells were incubated in
methionine/cysteine-free medium or phosphate-free medium (ICN) for
30 min and then exposed to [.sup.35S]-methionine and cysteine (66
uCi/ml; Tran.sup.35S-1abel, ICN) or [.sup.33P]-inorganic phosphate
(100 uCi/ml; ICN). Following a 2-h labeling period, cells were
harvested and cytoplasmic extracts prepared as previously
described.
EXAMPLE 4
[0049] Pulse-chase analysis of M2-1 protein
[0050] M2-1 protein maturation and stability were analyzed b y
metabolic labeling with a short exposure to [.sup.35S]-methionine
and cysteine (pulse) followed by varying incubation times
post-1abel (chase). HEp-2 cells infected with vTF7-3 were
transfected with plasmid encoding the wild-type M2-1 protein as
described above. Sixteen-hour post-transfection cells were
incubated in methionine/cysteine free medium for 30 min and then
exposed to [.sup.35S]-methionine and cysteine (100 uCi/ml;
Tran.sup.35S-1abel, ICN) for 1 5 min. Following the labeling
period, cells were either harvested and cytoplasmic extracts
prepared, or the label was removed, cells were washed and fresh
medium containing excess unlabeled methionine and cysteine (10 mM)
was added for 15-, 30-, or 60-min prior to harvest.
EXAMPLE 5
[0051] Immunoprecipitations of labeled proteins
[0052] Immunoprecipitation of RS virus-specific proteins from
cytoplasmic extracts was performed using an M2-1 protein-specific
monoclonal antibody (MAb), 5H5 or IC13 (the kind gift from G.
Toms), or a polyclonal anti-RS virus serum (Chemicon International)
and protein-G Sepharose (Pharmacia Biotech). Immunoprecipitated
proteins were analyzed by SDS-PAGE in 11% polyacrylamide gels under
reducing conditions and detected by fluorography (2, 17).
EXAMPLE 6
[0053] Effect of mutations in the Cys.sub.3--His.sub.1 motif of
M2-1 protein on RS virus transcription
[0054] The M2-1 protein causes a decrease in the efficiency of
transcription termination at RS virus gene junctions resulting in
an increased production of readthrough transcripts, thus
functioning as a transcription antiterminator (8, 13). Sequence
analysis revealed that the M2-1 protein contains a
Cys.sub.3--His.sub.1 amino acid sequence motif
(C--X.sub.7--C--X.sub.5--C--X.sub.3--H). A similar motif has been
shown to bind zinc i n Nup475 protein, a mammalian transcription
factor (36). To determine whether this motif was important for the
M2-1 protein to function as an antiterminator during viral
transcription, point mutations were generated in the cDNA encoding
the M2-1 protein (FIG. 1A). Three of the residues predicted to
coordinate a zinc ion, the cysteine residues at positions 7 and 15,
and the histidine a t position 25, were each changed to serines.
The mutants were named according to the following terminology: C7S,
C15S and H25S. In addition three other residues within the motif
which would not b e predicted to be involved in coordinating a zinc
ion were mutated as controls: K8Q, F9L, F9S, E10G and E10Q (FIG.
1A). The mutant M2-1 ORFs were cloned behind the T7 promoter in
pGEM3.
[0055] The effects of mutations in the Cys.sub.3--His.sub.1 motif
of the M2-1 protein on RS virus transcription were assayed using an
RS virus subgenomic replicon supported by the recombinant vaccinia
virus-T7 expression system (38). An RS virus subgenomic replicon
containing two genes separated by the M/SH gene junction (FIG. 1B)
was expressed from CDNA in cells also expressing the N, P, L and
M2-1 proteins from T7 expression plasmids. The construction of this
subgenomic replicon has been described previously (13). The effect
of the wild type and mutant M2-1 proteins on RS virus transcription
was determined by direct metabolic labeling of RNA. The synthesis
of discrete monocistronic mRNA1 and mRNA2 (FIG. 1B) was analyzed in
comparison to the synthesis of the dicistronic mRNA, r/t B,
generated by the failure of the polymerase to terminate
transcription at the end of mRNA1. The wild-type M2-1 protein
decreased transcriptional termination and increased readthrough
transcription as previously reported (11, 13). The increase in
readthrough transcription can be seen by comparing lanes 1 and 2 of
FIG. 1C. In the absence of M2-1, primarily the products of
replication and mRNA1, mRNA2, and a small amount of readthrough
from mRNA2 into trailer (r/t A) were synthesized. In the presence
of M2-1, a significant increase in the products of readthrough
transcription (polycistronic mRNAs) occurred, shown most strikingly
by the presence of r/t B (a dicistronic mRNA consisting of the
sequences of mRNAl and mRNA2). Detailed identification of the RNAs
in FIG. 1C has been presented in previous work (13). The data in
FIG. 1C show that the M2 proteins in which residues which are not
predicted to coordinate the binding of a zinc ion were changed
(K8Q, F9L, F9S, E10G, and E10Q) functioned similar to the wt M2-1
protein in causing an increase in readthrough transcription (FIG.
1C, lanes 2, 4, 5, 6, 7, and 8). However, mutations of the residues
of M2-1 which are predicted to coordinate the binding of a zinc ion
inhibited the ability of the protein from increasing readthrough
transcription (FIG. 1C, lanes 3, 9, and 10). Thus, the integrity of
the Cys.sub.3--His.sub.1 motif was important for maintaining the
function of the M2-1 protein as an antiterminator during RS virus
transcription.
EXAMPLE 7
[0056] Effects of mutations in the Cys--His.sub.1 motif on M2-1
protein mobility in SDS-PAGE
[0057] Expression of each of the mutant M2-1 proteins was analyzed
by immunoprecipitation followed by reducing SDS PAGE to test
whether each of the mutant proteins was expressed and stable. We
also examined the effects of the mutations on electrophoretic
mobility as previous reports showed the M2 protein migrated as a
doublet (27). The wild type and mutant M2-1 proteins were expressed
in HEp-2 cells from cDNAs using the recombinant vaccinia virus T7
expression system. Proteins were labeled with [.sup.35S]-methionine
and cysteine and immunoprecipitated from cell lysates using an
M2-specific MAb, 5H5. Immunoprecipitated proteins were analyzed by
SDS-PAGE under reducing conditions. In FIG. 2, it can b e seen that
specific mutations affected the mobility of the M2 protein. The
wild-type protein migrated as a doublet, with the majority of the
protein present as the slower migrating form (FIG. 2, lane 3). This
was also true of K8Q, F9L, F9S and E10Q (FIG. 2, lanes 5,6,7 and
9). The mutation E10G caused a shift in the distribution of protein
from the slower to the faster migrating form and a slight increase
in mobility (FIG. 2, lane 8). Mutations of the residues predicted
to coordinate the binding of zinc (C7S, C15S, and H25S) caused a
significant shift from the slower to the faster migrating form of
the protein (FIG. 2, lanes 4, 10, and 11). Thus, mutations in this
region had a significant effect on the electrophoretic mobility of
the M2-1 protein specifically altering the distribution of protein
between a slower and faster migrating form.
[0058] The nature of the two forms of the M2 protein, seen as a
doublet in reducing SDS-PAGE was investigated by metabolic pulse
labeling with [.sup.35S]-methionine and cysteine, followed by a
chase with excess unlabeled amino acids. The faster migrating form
of M2-1 was labeled initially (FIG. 3, lane 2) and then over time
chased into the slower migrating form (FIG. 3, lanes 3, 4 and 5).
These results suggest that the slower migrating form was a
post-translationally modified form of the M2-1 protein (FIG.
3).
EXAMPLE 8
[0059] Phosphorylation of M2-1 protein
[0060] The M2-1 protein has previously been reported to be
phosphorylated in RS virus-infected cells (18). Results of the
above chase analysis (FIG. 3) suggested that the cause of the
difference in mobility of the two forms of M2-1 was due to a
post-translational modification. Therefore, studies were performed
to investigate whether the mobility differences of the M2-1 protein
in SDS-PAGE was related to its phosphorylation state. Wild-type
M2-1 and mutant M2-1 proteins, C7S and E10G, were expressed in
vTF7-3 infected cells and labeled with [.sup.35S]-methionine and
cysteine, or with [.sup.33P]-inorganic phosphate. Proteins
synthesized in RS virus-infected cells were labeled under the same
conditions. Labeled proteins were immunoprecipitated with MAb 5H5
or polyclonal antiserum to RS virus and analyzed by SDS-PAGE under
reducing conditions.
[0061] Analysis of the [.sup.35S]-1abeled M2-1 protein produced in
RS virus-infected cells showed it was present as a doublet with the
majority of the protein (approximately 75%) found in the faster
migrating form (FIG. 4, lanes 11 and 13). When labeled with
[.sup.33P]-inorganic phosphate, only a single labeled band was
observed which corresponded to the slower migrating form of M2-1
(FIG. 4, lanes 10 and 12). Expression of the wild-type M2-1 protein
alone from a plasmid in cells also showed that it was the slower
form of M2-1 that was phosphorylated, whereas the faster form was
not (FIG. 4, lanes 4 and 5). This demonstrated that the M2-1
protein could b e phosphorylated in the absence of other RS virus
proteins. In contrast, the majority of the mutant C7S M2-1 protein
migrated as the faster form which was not detectably phosphorylated
(FIG. 4, lanes 6 and 7). Analysis of the EIOG M2-1 mutant protein
showed a shift in the distribution towards the faster migrating
form which corresponded with an approximately 50% decrease in
phosphorylation compared to wt M2-1 protein (FIG. 4, lanes 8 and
9).
[0062] These results showed that the faster and slower migrating
forms of M2-1 protein observed by SDS-PAGE under reducing
conditions were differentiated by their phosphorylation state; the
slower migrating form was phosphorylated and the faster migrating
form was not. In addition, the mutations of the predicted zinc
coordinating residues in the Cys.sub.3--His.sub.1 motif prevented
efficient phosphorylation of the M2-1 protein. Treatment of the
wild type M2-1 protein with calf alkaline phosphatase (CIP)
confirmed these results as digestion with CIP resulted in a shift
of the slower migrating form to the faster form (T. Cartee,
unpublished data).
EXAMPLE 9
[0063] Effects of mutations in Cys.sub.3--His.sub.1 motif of M2-1
on the interaction with N protein
[0064] The M2-1 and N proteins have been demonstrated to interact
in RS virus infected cells and when the two proteins are
co-expressed (9). The effect of mutations in the
Cys.sub.3--His.sub.1 motif of the M2-1 protein on its interaction
with the N protein was assayed by coexpression of wild type and
mutant M2-1 proteins with the N protein in HEp-2 cells. Proteins
labeled with [.sup.35S]-methionine and cysteine were
immunoprecipitated from cell lysates using the M2-1 specific MAb,
5H5, and analyzed by SDS-PAGE under reducing conditions. The N
protein was coprecipitated with the wild-type and F9L M2-1 proteins
(FIG. 5, lanes 2 and 4). Mutation of the cysteine residues at
positions 7 and 15 (FIG. 5, lanes 3 and 6) and the histidine at
position 25 (data not shown) of the M2-1 protein severely decreased
the efficiency with which N protein was coprecipitated,
demonstrating the importance of the predicted zinc-coordinating
residues for maintenance of the M2-1 interaction with N protein.
Surprisingly, the E10G mutation, which did not prevent M2-1 protein
mediated antitermination during RS virus transcription, prevented
the M2-1 protein from interacting with the N protein. The reason
for this is not clear, but may be related to the presence of a
glycine residue rather than the loss of the glutamate as the EIOQ
mutation in the M2-1 protein did interact with the N protein (data
not shown).
[0065] These results demonstrate the requirement for maintaining
the Cys.sub.3--His.sub.1 motif in order for M2-1 protein to
interact with N protein. In addition, the phenotype of the EIOG
mutation suggested that the interaction is specific in that it can
be disrupted by a single mutation in a non-coordinating residue of
the predicted zinc-binding domain. This mutation separates the
function of M2-1 protein in transcription from its ability to
interact with N protein, implying that this interaction is not
required for M2 to function during transcription. However, the
conditions under which the immunoprecipitations were performed were
stringent and may disrupt weak interactions which could be
functionally relevant in cells. Additionally it should be noted
that these results do not rule out the possibility that this
interaction is mediated via another molecule with which both N and
M2-1 proteins interact.
EXAMPLE 10
[0066] Requirement of individual RS virus protein for RNA
transcription and replication
[0067] The requirement for individual RS virus proteins in order to
obtain RNA transcription and replication was examined. Cells were
infected with VVT7 and transfected with plasmids expressing a
subgenomic RS virus RNA replicon, pWT5 (FIG. 6), and, in various
combinations, individual plasmids expressing the RS virus core
polymerase protein (L), the nucleocapsid protein (N), the
phosphoprotein (P) or M2-1 protein.
[0068] As can be seen in FIG. 7 lane 4, cells transfected with all
5 plasmids synthesized the genomic positive and negative strand RNA
products of replication and the single mRNA product of
transcription as well as a transcriptive product of readthrough
from failing to terminate at the end of the mRNA and reading
through into the trailer gene. Omission of the N, P or L expression
plasmids individually (lanes 3, 5, or 1 respectively) resulted in
failure of any RNAs to be synthesized. Omission of the M2-1
expression plasmid (lane 2) resulted in failure to synthesize the
transcriptive readthrough RNA. Thus these data showed that the N, P
and L genes are sufficient for RNA replication, but the M2-1
protein provides an additional function for transcription. It
serves as a transcription anti terminator.
[0069] Discussion
[0070] Sequence analysis of the RS virus M2-1 protein revealed a
Cys.sub.3--His.sub.1 motif which had been shown to bind zinc in
another protein (5, 36). Mutational analysis of the
Cys.sub.3--His.sub.1 motif demonstrated that maintaining the
cysteine and histidine residues predicted to coordinate zinc was
essential for the functional integrity of the M2-1 protein.
Alteration of the predicted zinc-coordinating residues resulted in
an M2-1 protein which was unable to enhance transcriptional
readthrough at RS virus gene ends. In addition, mutations of the
predicted zinc coordinating residues resulted in an alteration in
the migration pattern of the M2-1 protein in SDS PAGE from a
relatively slow to a relatively fast migrating form. The two forms
of M2-1 protein differed in phosphorylation, the slower form being
phosphorylated and the faster form not.
[0071] Mutation of the predicted zinc coordinating residues also
inhibited the interaction of the M2-1 protein with the N protein.
Mutating any one of the residues of the M2-1 protein predicted to
coordinate zinc resulted in the same phenotype, whereas mutating
other non-coordinating residues in the Cys.sub.3--His.sub.1 motif
had little if any effect on the antitermination function of the
M2-1 protein. Thus maintaining the potential zinc coordinating
residues of the Cys.sub.3--His.sub.1 motif was essential for M2-1
function. These results are consistent with the idea that the
Cys.sub.3--His.sub.1 motif coordinates the binding of an ion of
zinc. Studies are underway to directly demonstrate that M2-1 binds
zinc using various techniques.
[0072] The data presented here show that the two forms of M2-1
protein separated by SDS-PAGE under reducing conditions are
discriminated by whether or not they are phosphorylated. The slower
migrating form of M2-1 was phosphorylated, whereas the faster form
was not. Multiple forms of the M2-1 protein can be seen in FIG. 2
and 3, two major bands and at least two other minor species. These
minor species may correspond to those observed by Routledge et al.
in infected cells (27). Additionally, the extent to which the M2-1
protein is phosphorylated has not yet been determined and the less
prevalent species may represent differentially phosphorylated forms
of M2-1.
[0073] The integrity of the Cys.sub.3--His.sub.1 motif was
important for phosphorylation. Protein folding studies have shown
that zinc binding and protein folding are tightly coupled with zinc
binding conferring structural stability (6). One could hypothesize
that M2-1 which has zinc bound is folded and can be recognized by
the appropriate cellular kinase, whereas mutants which prevent the
binding of zinc would not be properly folded and would not b e
recognized by the kinase. Such a model would mean that zinc binding
is essential for phosphorylation, which, in turn, may play a role
in M2-1 function. In such a scenario, zinc binding could play a
crucial role in the regulation of RS virus transcription.
[0074] The role of the M2-1:N protein-protein interaction in the RS
virus replication cycle is currently unknown. The EIOG M2-1 protein
is active in transcription, but results reported above indicate
that it does not interact with the N protein, suggesting that this
interaction is not required for M2-1 to enhance transcriptional
readthrough. This interaction may be important at another point
during virus replication. The M2-1 protein was initially
characterized as a matrix-like protein as it dissociated from
nucleocapsids under similar conditions to the matrix protein, M
(14). If M2-1 can function as a matrix like protein, in addition to
its activity during transcription, it is possible that the
interaction between M2-1 and N may be involved in virus
assembly.
[0075] Zinc-binding motifs have been found in a number of proteins
and, in many cases, mediate protein-protein or protein-nucleic acid
interactions (1, 21, 22, 23, 30, 31, 32). Often the binding of zinc
plays a purely structural role holding the protein in a
conformation which is functional (24). It is hypothesized that the
Cys.sub.3--His.sub.1 motif of M2-1 binds zinc in order for the
protein to be in a conformation which allows it to function in RS
virus transcription, to interact with the N protein, and to be
efficiently phosphorylated. In conclusion, the data presented above
demonstrated that the predicted zinc-binding motif of M2-1 is
essential for maintaining the functional integrity of the protein,
and that there are at least two forms of M2-1 produced in infected
cells which can be distinguished by their phosphorylation
state.
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[0116] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0117] 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. The present examples, along
with the methods, procedures, treatments, molecules, and specific
compounds described herein are presently representative of
preferred embodiments, are exemplary, and are not intended as
limitations on the 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
10 1 19 PRT Unknown Domain 2..8, 10..14 and 16..18 Cys3-His1 zinc
binding domain consensus sequence, Xaa at any position may be any
amino acid 1 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys 5 10 15 Xaa Xaa Xaa His 2 30 PRT Artificial Sequence
wild-type M2 protein from respiratory syncytial virus 2 Met Ser Arg
Arg Asn Pro Cys Lys Phe Glu Ile Arg Gly His Cys 5 10 15 Leu Asn Gly
Lys Arg Cys His Phe Ser His Asn Tyr Phe Glu Trp 20 25 30 3 30 PRT
Artificial Sequence mutant M2 protein from respiratory syncytial
virus 3 Met Ser Arg Arg Asn Pro Ser Lys Phe Glu Ile Arg Gly His Cys
5 10 15 Leu Asn Gly Lys Arg Cys His Phe Ser His Asn Tyr Phe Glu Trp
20 25 30 4 30 PRT Artificial Sequence mutant M2 protein from
respiratory syncytial virus 4 Met Ser Arg Arg Asn Pro Cys Gln Phe
Glu Ile Arg Gly His Cys 5 10 15 Leu Asn Gly Lys Arg Cys His Phe Ser
His Asn Tyr Phe Glu Trp 20 25 30 5 30 PRT Artificial Sequence
mutant M2 protein from respiratory syncytial virus 5 Met Ser Arg
Arg Asn Pro Cys Lys Leu Glu Ile Arg Gly His Cys 5 10 15 Leu Asn Gly
Lys Arg Cys His Phe Ser His Asn Tyr Phe Glu Trp 20 25 30 6 30 PRT
Artificial Sequence mutant M2 protein from respiratory syncytial
virus 6 Met Ser Arg Arg Asn Pro Cys Lys Ser Glu Ile Arg Gly His Cys
5 10 15 Leu Asn Gly Lys Arg Cys His Phe Ser His Asn Tyr Phe Glu Trp
20 25 30 7 30 PRT Artificial Sequence mutant M2 protein from
respiratory syncytial virus 7 Met Ser Arg Arg Asn Pro Cys Lys Phe
Gly Ile Arg Gly His Cys 5 10 15 Leu Asn Gly Lys Arg Cys His Phe Ser
His Asn Tyr Phe Glu Trp 20 25 30 8 30 PRT Artificial Sequence
mutant M2 protein from respiratory syncytial virus 8 Met Ser Arg
Arg Asn Pro Cys Lys Phe Gln Ile Arg Gly His Cys 5 10 15 Leu Asn Gly
Lys Arg Cys His Phe Ser His Asn Tyr Phe Glu Trp 20 25 30 9 30 PRT
Artificial Sequence mutant M2 protein from respiratory syncytial
virus 9 Met Ser Arg Arg Asn Pro Cys Lys Phe Glu Ile Arg Gly His Ser
5 10 15 Leu Asn Gly Lys Arg Cys His Phe Ser His Asn Tyr Phe Glu Trp
20 25 30 10 30 PRT Artificial Sequence mutant M2 protein from
respiratory syncytial virus 10 Met Ser Arg Arg Asn Pro Cys Lys Phe
Glu Ile Arg Gly His Cys 5 10 15 Leu Asn Gly Lys Arg Cys His Phe Ser
Ser Asn Tyr Phe Glu Trp 20 25 30
* * * * *