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Journal of Virology, December 1999, p. 10519-10524, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Open Reading Frame 57 Gene Product of
Herpesvirus Saimiri Shuttles between the Nucleus and Cytoplasm and
Is Involved in Viral RNA Nuclear Export
Delyth J.
Goodwin,
Kersten T.
Hall,
Alex J.
Stevenson,
Alex
F.
Markham, and
Adrian
Whitehouse*
Molecular Medicine Unit, University of Leeds,
St. James's University Hospital, Leeds LS9 7TF, United Kingdom
Received 7 June 1999/Accepted 20 August 1999
 |
ABSTRACT |
The herpesvirus saimiri open reading frame (ORF) 57 is homologous
to genes identified in all classes of herpesviruses. It has previously
been shown to regulate gene expression through a posttranscriptional
mechanism. We demonstrate in this report that the expression of the ORF
57 protein leads to the cytoplasmic accumulation of glycoprotein B and
capsid mRNAs. We also demonstrate that ORF 57 has the ability to
specifically bind viral RNA transcripts. Utilizing an interspecies
heterokaryon assay, we show that ORF 57 has the ability to shuttle
between the nucleus and the cytoplasm. Furthermore, we show that ORF 57 contains a relatively leucine-rich sequence which shares some homology
with nuclear export signals (NES) found in a number of proteins with
the ability to shuttle between the nucleus and the cytoplasm. Moreover,
we demonstrate that the ORF 57 NES enables the nuclear export of a
heterologous protein and that mutation of the conserved leucine
residues contained within the ORF 57 NES signal abrogates the ability
of the ORF 57 protein to shuttle between the nucleus and cytoplasm.
These results suggest that ORF 57 is involved in mediating the nuclear export of viral transcripts.
| |
TEXT |
Herpesvirus saimiri (HVS) is a
lymphotrophic herpesvirus of squirrel monkeys (Saimiri
sciureus), which persistently infects its natural host
asymptomatically but which can cause fatal T-cell lymphomas and
lymphoproliferative diseases in other species of New World primates
(9). Analysis of the HVS (strain A11) genome indicates that
it shares significant homology, at a colinear level, with the
gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's
sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (1,
28, 34). Gene expression during the HVS lytic replication cycle
is regulated by the products of the two major transcriptional regulating genes containing the open reading frames (ORFs) 50 and 57 (22, 23, 38). ORF 50 produces two gene products which are
homologous to the EBV BRLF1 protein (Rta protein) (22, 36) and function as sequence-specific transactivators (35). ORF 57 is homologous to genes identified in all classes of herpesviruses, including the EBV Mta protein transactivator encoded by BMLF1 and IE63
or ICP27 of herpes simplex virus type 1 (HSV-1) (1, 15, 22).
The ORF 57 gene product is a 52-kDa multifunctional protein. We have
previously shown that transactivation of late viral genes by ORF 57 occurs at a posttranscriptional level, whereas repression of gene
expression appears to correlate with the presence of introns (37,
38). In addition, ORF 57 is responsible for the redistribution of
the U2 and SC-35 splicing factors during an HVS infection
(6). This suggests that ORF 57 plays a role in RNA
processing and is functionally homologous to the more widely studied
HSV-1 IE63 protein (30, 37). In addition to the above
properties, the more widely characterized IE63 protein also contributes
to the shutoff of host cell protein synthesis and to a decrease in
cellular mRNA levels during infection. Infections utilizing IE63
deletion mutants result in increased levels of cellular protein
synthesis and mRNA levels compared to wild-type infections (11,
12).
Analysis of IE63 has shown that it contains many functional domains,
including an RGG box required for RNA binding (19), an
N-terminal nuclear localization signal (NLS) (13, 18), and
C-terminal transactivation and repression domains (31). Recent analysis has shown it also expresses a leucine-rich nuclear export signal (NES) that is homologous to sequences found in a number
of shuttle proteins, including human immunodeficiency virus type 1 (HIV-1) Rev (14, 21). Further analysis of IE63 has shown
that it has the ability to shuttle between the nucleus and the
cytoplasm (20, 25), thus promoting the nuclear export of
HSV-1 intronless RNAs (29, 33).
In this report, we further investigate the role of the HVS ORF 57 protein. We demonstrate that the ORF 57 protein is required for the
cytoplasmic accumulation of virus mRNA. In addition, we show that ORF
57 has the ability to shuttle between the nucleus and the cytoplasm.
Furthermore, we demonstrate that ORF 57 encodes an NES which enables
the rapid nuclear export of a heterologous protein. These results
suggest that ORF 57 plays a role in mediating the nuclear export of
viral transcripts.
ORF 57 increases cytoplasmic transport of viral mRNA.
It has
been previously shown that ORF 57 transactivates a range of HVS
promoters, including glycoprotein B (gB) and capsid, but does not
significantly alter the level of mRNA, suggesting that ORF 57 acts via
a posttranscriptional mechanism (37). In order to determine
if ORF 57 affects the nuclear cytoplasmic transport of viral mRNAs,
Northern blot analysis was performed. To assess the effect of ORF 57 on
gB mRNA, a transfer vector containing the full-length coding region and
the promoter of gB was constructed. This region was PCR amplified using
the primers 5'-GCG GGA TCC GTT ACA TGA TGC GCA TGC TAG and 5'-GCG GGA
TCC CCA TGT CAA GAC AGC AAC TC. These oligonucleotides incorporated
BamHI restriction sites for convenient cloning of the
PCR product. This fragment was inserted into the polylinker region of
pUC18 to derive pUCgB. In addition, to assess the effect of ORF 57 on
capsid mRNA a transfer vector, pEcoB, containing the
full-length coding region and promoter of the capsid gene was
also utilized (16). Total, nuclear, and cytoplasmic
RNA was then isolated separately from Cos-7 cells transfected with
pUCgB or pEcoB in the absence or the presence of
pRSVORF57, a eukaryotic expression vector expressing the ORF 57 coding
region (37). The RNA was then separated by electrophoresis on a 1% denaturing formaldehyde agarose gel, transferred to Hybond-N membranes, and hybridized with 32P-radiolabelled random
primed probes specific for the HVS gB or the capsid coding sequences
(Fig. 1). The results of the Northern blot analysis suggest that ORF 57 does not affect the quantities of gB
or capsid transcripts but is required for the efficient cytoplasmic
accumulation of gB and capsid transcripts.
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FIG. 1.
ORF 57 increases cytoplasmic transport of viral mRNA.
Cos-7 cells transfected with pUCgB (a) and pEcoB (b) in the
absence or presence of pRSVORF57. Total (lanes 1 and 2), nuclear (lanes
3 and 4), and cytoplasmic (lanes 5 and 6) RNA was then isolated and
separated by elecrophoresis on a 1% denaturing formaldehyde agarose
gel. The RNA was transferred to Hybond-N membranes and hybridized with
32P-radiolabelled randomly primed probes specific for the
HVS gB and the capsid coding sequence.
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The ORF 57 protein binds viral mRNA.
In order to determine
whether ORF 57 binds viral RNA, Northwestern analysis was performed.
Recombinant ORF 57 protein was produced and purified as an
amino-terminal glutathione S-transferase (GST) fusion
protein. A PCR product of the coding region of ORF 57 containing 78,394 to 79,684 bp of the previously published sequence (1) was
generated by PCR, using the primers 5'-CGC GGA TCC GGA ATT TCA TCA GAT
GAT GAC and 5'-GCG GGA TCC CTG AGT AGG TAA GAA AAA CAG CCC; these
oligonucleotides incorporated BamHI restriction sites to
facilitate subcloning into the expression vector, pGEX-2T (Pharmacia
Biotech), thus deriving pGEX57. The ORF 57 fragment was expressed as a
GST fusion protein in Escherichia coli DH5
and purified
from the crude lysate by incubation with glutathione-Sepharose 4B
affinity beads according to the manufacturer's specification
(Pharmacia Biotech). The molecular mass of the GST-ORF 57 fusion
protein was predicted to be 81 kDa. However, analysis demonstrated that
the expressed GST-ORF 57 protein produced was a stable breakdown
product containing the amino-terminal portion of the protein of ca. 55 kDa (Fig. 2a). This smaller product may have been due to premature translational termination; however, DNA
sequencing confirmed the integrity of the DNA sequence, further suggesting a stable breakdown product. In order to determine whether this expressed protein had the ability to bind RNA, the purified GST-ORF 57 recombinant protein and a control, GST alone, were separated
on a sodium dodecyl sulfate (SDS)-12% polyacrylamide gel and
transferred onto nitrocellulose (Amersham). The nitrocellulose-bound proteins were denatured by the addition of 6 M guanidine-HCl in binding
buffer (100 mM Tris-HCl [pH 8.0], 50 mM NaCl, 1 mM EDTA) for 10 min.
The proteins were then renatured by using six 50% stepwise dilutions
of the guanidine-HCl in binding buffer. The filter was rinsed in
binding buffer and blocked by preincubation in 2% (wt/vol) nonfat milk
powder-1 mM dithiothreitol for 2 h at 20°C. The filter-bound
protein was incubated with 32P-radiolabelled RNA probes
specific for HVS gB or actin. The gB RNA probe was synthesized by in
vitro transcription of a PCR product containing a T7 transcription
start site in the 5' primer in the presence of [32P]UTP
(Ambion). The gB coding region was amplified with primers 5'-TAA TAC
GAC TCA CTA TAG GGA ATG GTA CCT AAT AAA CAC TTA CTG and 5'-TGA ACT GCA
CAG ATC CTA TAT. The actin probe was generated using the control
vector, pTriplescript, according to the manufacturer's directions
(Maxiscript Kit; Ambion). The labelled probes were hybridized with the
filter-bound ORF 57 protein for 16 h at 20°C. The results
demonstrate that the amino-terminal portion of ORF 57 can specifically
bind the viral gB RNA transcripts but did not interact with the control
cellular RNA (Fig. 2b).
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FIG. 2.
The ORF 57 protein binds viral mRNA. (a) Coomassie
blue-stained gel of GST (lane 1) and GST-ORF 57 (lane 2) proteins
expressed in E. coli DH5 and purified from the crude
lysate by incubation with glutathione-Sepharose 4B beads according to
manufacturer's specifications. (b) RNA-binding assay of the ORF 57 protein. GST and GST-ORF 57 proteins were separated on an SDS-12%
polyacrylamide gel and transferred onto nitrocellulose (Amersham). The
nitrocellulose bound proteins were denatured by using 6 M guanidine-HCl
and then renatured by using six 50% stepwise dilutions of the
guanidine-HCl in binding buffer. The filter-bound ORF 57 protein was
then hybridized with 32P-radiolabelled RNA probes specific
for HVS gB (i) and actin (ii).
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The ORF 57 protein shuttles between the nucleus and cytoplasm.
In order to determine whether the ORF 57 protein shuttles between the
nucleus and cytoplasm, an interspecies heterokaryon assay was utilized
(3, 20, 26). Cos-7 cells seeded at 2 × 105
cells per 35-mm-diameter petri dish were transiently transfected with 2 µg of pRSVORF57. After 18 h, mouse 3T3 cells (5 × 105 cells/well) were plated onto the Cos-7 cells in medium
containing 50 µg of cycloheximide per ml. Four hours later the cells
were washed in phosphate-buffered saline (PBS) and fused by the
addition of 2 ml of 50% polyethylene glycol (wt/wt) in PBS. After 2 min the cells were washed extensively in PBS. After this washing, the
cells were returned to medium containing 50 µg of cycloheximide per
ml for 60 min. Cells were then fixed with 4% formaldehyde in PBS,
washed in PBS three times, and permeabilized in 0.5% Triton X-100 for
5 min. The cells were rinsed in PBS and blocked by preincubation with
1% (wt/vol) nonfat milk powder for 1 h at 37°C. A 1:100
dilution of ORF 57 antibody (27) was layered over the cells
and incubated for 1 h at 37°C. Fluorescence-conjugated
anti-mouse immunoglobulin (Dako) at a 1:50 dilution and a costain of
0.5 µg of Hoechst dye (Sigma) per ml were added for 1 h at
37°C. After each incubation step, cells were washed extensively with
PBS. Hoechst dye allowed the differentiation between monkey and mouse
nuclei. As previously reported (20), monkey cells stained
diffusely throughout the nuclei, whereas mouse nuclei stained with a
distinctive speckled pattern (Fig. 3a).
The immune fluorescence slides were observed by using a Zeiss Axiovert
135TV inverted microscope with a Neofluar 40× oil immersion lens.
Analysis of the interspecies heterokaryons demonstrated that ORF 57 was
expressed in both monkey and mouse cell nuclei. This indicates that ORF
57 in transfected cells is able to shuttle between the nucleus and
cytoplasm (Fig. 3b).
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FIG. 3.
(a) Hoechst dye staining of monkey and mouse cell
nuclei. Hoechst dye allowed differentiation between monkey (i) and
mouse (ii) nuclei. Monkey cells stained diffusely throughout the
nuclei, whereas mouse nuclei stained with a distinctive speckled
pattern. (b) The ORF 57 protein shuttles between the nucleus and
cytoplasm. Cos-7 cells seeded at 2 × 105 cells per
35-mm-diameter petri dish were transiently transfected with 2 µg of
pRSVORF57. After 18 h, mouse 3T3 cells (5 × 105
cells/well) were plated onto the Cos-7 cells in medium containing 50 µg of cycloheximide per ml. Four hours later the cells were washed in
PBS and fused by the addition of 2 ml of 50% polyethylene glycol
(wt/wt) in PBS. After being washed, the cells were returned to medium
containing 50 µg of cycloheximide per ml for 60 min. Cells were
incubated with a 1:100 dilution of ORF 57 antibody and then costained
with 0.5 µg of Hoechst dye (i) and fluorescein-conjugated anti-mouse
immunoglobulin (ii) per ml.
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The ORF 57 gene product expresses an NES.
In order for a
protein to shuttle between the nucleus and cytoplasm it requires both
an NLS and an NES. An increasing number of viral proteins, including
HIV-1 Rev, human T-cell lymphotropic virus type 1 Rex, HSV-1 IE63, and
EBV Mta, have been found to contain a leucine-rich NES sequence
enabling the protein to shuttle between the nucleus and cytoplasm
(2, 14, 20, 21, 25, 29, 33). Analysis of the ORF 57 coding
region identified a relatively leucine-rich region containing a
sequence of amino acids resembling the consensus NES sequence. In order
to determine whether the putative NES signal (ILPKSGEPKLFL)
expressed by ORF 57 enables nuclear export of a heterologous protein,
the sequence was fused with the green fluorescent protein (GFP).
Synthetic oligonucleotides encoding the putative ORF 57 NES
(nucleotides 78835 to 78870 of the published sequence) were
synthesized. These oligonucleotides incorporated XhoI and
BamHI restriction sites for convenient cloning. The
oligonucleotides were annealed and ligated with pEGFP-C1 (Clontech) to
create an in-frame carboxy-terminal fusion of the NES sequence and GFP,
yielding pEGFP-57NES. Cos-7 cell monolayers were transfected with 2 µg of either pEGFP-C1 or pEGFP-57NES, and the subcellular
localization of GFP was observed by fluorescence microscopy. Results
showed that cells transfected with pEGFP-C1 displayed a fluorescence
pattern throughout the cell in both the nucleus and cytoplasm. However,
the fluorescence pattern observed in pEGFP-57NES-transfected cells was
confined to the cytoplasm (Fig. 4),
suggesting that the ORF 57-expressed NES is sufficient to direct the
fusion protein from the nucleus to the cytoplasm.
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FIG. 4.
The ORF 57 gene product expresses a nuclear export
signal. Cos-7 cell monolayers were transfected with 2 µg of either
pEGFP-C1 (i) or pEGFP-57NES (ii). After 24 h, the subcellular
localization of GFP was observed by using fluorescence microscopy.
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The NES signal is required for ORF 57 to shuttle between the
nucleus and cytoplasm.
To further demonstrate that the NES
sequence enables the ORF 57 protein to shuttle between the nucleus and
the cytoplasm, site-directed mutagenesis of the NES was performed. The
ORF 57-NES mutant was generated by a PCR-based method which
incorporated the alteration of the conserved leucine residues at bp
78863 and 78869 of the published sequence. Two PCR products of the ORF
57 coding region containing the sequences bp 78291 to 78872 and bp 78872 to 79624 of the published sequence were generated by using the
primers NES1 (5'-CCC AAG CTT AAC TGC CCA ATT GGA AGA TAT AAT TG), NES2
(5'-CCC CCG CGG GCT TTT GGC TCT CCA GAT TTA GGC AA), NES3 (5'-CGC CCG
CGG GCC TGT ACC TTC GTT GCC TTG CCA A), and NES4 (5'-CCG CTC GAG CTG
AGT AGG TAA GAA AAA CAG CCC TGT). Primers NES1 and NES4 contained
HindIII and XhoI restriction sites to facilitate the subcloning into the expression vector pcDNA3.1 (Invitrogen), whereas NES2 and NES3 contained SstII
restriction sites to facilitate ligation of the PCR products but also
mutated the two conserved leucine residues contained in the ORF 57-NES sequence. The PCR products were ligated with pcDNA3.1 to derive the ORF
57-NES mutant, p57-NES. DNA sequencing was performed to confirm the
mutation of the specified residues (data not shown). In order to assess
the effect of the NES mutation on the ability of ORF 57 to shuttle
between the nucleus and the cytoplasm, a heterokaryon assay was
performed using p57-NES as previously described (Fig.
5). Analysis of heterokaryons which
contained the mutated ORF 57 demonstrated that this protein was only
expressed in the monkey nuclei. This indicates that mutation of the
conserved leucine residues contained within the NES sequence abrogated
the ability of the ORF 57 protein to shuttle between the nucleus and
the cytoplasm.
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FIG. 5.
Mutation of the ORF 57 NES abrogated the ability of the
ORF 57 protein to shuttle between the nucleus and cytoplasm. Cos-7
cells seeded at 2 × 105 cells per 35-mm-diameter
petri dish were transiently transfected with 2 µg of p57-NES. After
18 h, mouse 3T3 cells (5 × 105 cells/well) were
plated onto the Cos-7 cells in medium containing 50 µg of
cycloheximide per ml. Four hours later the cells were washed in PBS and
fused by the addition of 2 ml of 50% polyethylene glycol (wt/wt) in
PBS. After being washed, the cells were returned to medium containing
50 µg of cycloheximide per ml for 60 min. Cells were then incubated
with a 1:100 dilution of ORF 57 antibody and costained with 0.5 µg of
Hoechst dye (i) and fluorescein-conjugated anti-mouse immunoglobulin
(ii) per ml.
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We have previously demonstrated that the ORF 57 gene product
transactivates a range of HVS promoters but does not significantly
alter the level of mRNA, suggesting a posttranscriptional mechanism.
In
addition, the effect of ORF 57 is independent of either the
promoter
which drives transcription or the temporal class of this
promoter.
However, we were unable to determine the effect of ORF
57: whether it
affects the mRNA processing, transport, or translational
efficiency. In
this report, we demonstrate that the ORF 57 protein
is required for the
cytoplasmic accumulation of virus mRNA. In
addition, we show that ORF
57 can bind viral RNA and shuttle between
the nucleus and cytoplasm.
Furthermore, we demonstrate that ORF
57 expresses an NES which is
required for ORF 57's ability to
shuttle between the nucleus and the
cytoplasm and that it enables
the rapid nuclear export of a
heterologous protein. These results
suggest that ORF 57 mediates the
nuclear export of viral
transcripts.
ORF 57 is a virus-encoded nuclear cytoplasmic shuttle protein, as are
HIV-1 Rev, HTLV-1 Rex, HSV-1 IE63, EBV Mta, and adenovirus
E4 34-kDa
oncoprotein (
2,
7,
14,
20,
21,
25,
29,
32,
33). Probably the
best characterized of these proteins
is HIV-1 Rev, which is involved in
promoting the export of intron-containing
mRNAs, which is mediated
through the binding of the Rev protein
to the Rev response elements
(RRE) contained within the introns
of these mRNAs (
8).
Moreover, HSV-1 IE63 selectively binds
intronless RNAs, and export of
these intronless RNAs is greatly
reduced during IE63 mutant virus
infections, suggesting that IE63
mediates the export of intronless RNAs
(
29,
33). All of these
proteins contain leucine-rich NESs
that enable rapid nuclear export.
Similar sequences which function as
NESs have been identified
in a wide range of proteins, including PK1,
transcription factor
IIIA, the yeast protein Gle 1, and MDM2 (reviewed
in reference
17). Recently, CRM1 (chromosomal region
maintenance 1) or exportin
1, a protein that shares homology with
members of the importin-karyopherin
nuclear transport pathway, has been
identified as a nuclear export
receptor for proteins carrying a
leucine-rich NES in a process
that also requires the GTP-bound form of
Ran (
10,
24). Furthermore,
exportin 1 has been shown to
interact with nuclear pore complex
proteins, namely, the nucleoporins
CAN/Nup214 and Nup88 (
24),
suggesting that exportin 1 is the
bridging protein for the interactions
of NES-containing proteins and
the nuclear pore complex. Recent
analysis has demonstrated that
exportin 1 mediates the function
and intracelluar localization of the
EBV Mta protein (
4). It
will be of interest to determine
whether ORF 57 interacts with
any components of this cellular
pathway.
In addition, virally encoded nuclear cytoplasmic shuttle proteins have
the ability to bind RNA. As previously mentioned, the
HIV-1 Rev protein
binds to the RRE contained within the introns
of these mRNAs mediated
by an arginine-rich region (
8). Moreover,
RNA binding by
HSV-1 IE63 is mediated by an arginine- and glycine-rich
sequence
resembling an RGG box motif, a putative RNA-binding determinant
found
in a number of cellular nuclear proteins involved in mRNA
and rRNA
metabolism (
19). However, ORF 57 and other IE63 homologues,
including EBV Mta, do not contain a homologous arginine- and
glycine-rich
RGG box motif. Recent analysis of the EBV Mta protein has
suggested
two regions which may contain alternative RNA-binding
determinants.
The EBV Mta and ORF 57 both have relatively arginine-rich
amino
termini. RNA-binding analysis of an EBV Mta-GST fusion product
suggested that this arginine-rich region in the amino terminus
of Mta
is likely to mediate RNA binding (
32). In addition, EBV
Mta
also contains an Arg-X-Pro tripeptide repeat homologous to
an
RNA-binding determinant in the HSV-1 US11 protein. However,
deletion
mutants of Mta have demonstrated this was not required
for RNA binding
(
5). Analysis of the ORF 57 coding region shows
this protein
does not express the Arg-X-Pro RNA-binding domain.
Nevertheless,
data described in this report, utilizing the GST-ORF
57 fusion protein,
suggest that the amino terminus of ORF 57 contains
the RNA-binding
determinant. However, it cannot be excluded that
the degraded
carboxy-terminal portion of the ORF 57 protein may
also include
RNA-binding determinants. Therefore, further analysis
is now required
to identify the functional domain(s) contained
within ORF 57 which are
responsible for RNA
binding.
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ACKNOWLEDGMENTS |
This work was supported in part from grants from the Medical
Research Council (MRC) and Yorkshire Cancer Research. A.W. and D.J.G.
are recipients of an MRC fellowship and an MRC studentship, respectively.
We thank Rick Randall for providing the SB monoclonal antibody.
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FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Medicine Unit, University of Leeds, St. James University Hospital,
Leeds LS9 7TF, United Kingdom. Phone: 44-(0)113-2066328. Fax:
44-(0)113-2444475. E-mail: A.Whitehouse{at}leeds.ac.uk.
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Journal of Virology, December 1999, p. 10519-10524, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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