Department of Microbiology and Immunology (M/C 790), College of Medicine, The University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, IL 60612, USA
Correspondence
Bin He
tshuo{at}uic.edu
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ABSTRACT |
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INTRODUCTION |
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The 134·5 protein of HSV-1 consists of a large amino-terminal domain, a linker region of triplet repeats (AlaThrPro) and a carboxyl-terminal domain (Chou & Roizman, 1990
). The amino-terminal domain of the protein has 150 residues that facilitate virus egress, although the underlying mechanism remains unknown (Jing et al., 2004
). The carboxyl-terminal domain functions to prevent the PKR response in virus infection (Cheng et al., 2001a
; Chou & Roizman, 1994
; He et al., 1996
). This portion of the protein is similar to the corresponding domain of the cellular protein GADD34 expressed under conditions of DNA damage, growth arrest, differentiation and apoptosis (Hollander et al., 1997
; Lord et al., 1990
; Zhan et al., 1994
). The linker region containing the triplet repeats is unique to the
134·5 protein encoded by HSV-1, but the number of repeats varies from strain to strain (Bower et al., 1999
; Chou & Roizman, 1990
; Perng et al., 2002
). Experiments suggest that the triplet repeats determine virus invasion of the central nervous system from the peripheral tissue (Bower et al., 1999
; Mao & Rosenthal, 2003
; Perng et al., 2002
). These observations are consistent with the notion that variation in the triplet repeats serves to regulate the functions of the
134·5 protein. However, the triplet repeats are not present in the
134·5 protein encoded by HSV-2 (McGeoch et al., 1991
).
Recent studies have demonstrated that variation in the triplet repeats seems to affect distribution of the 134·5 protein in transfected mammalian cells (Mao & Rosenthal, 2002
). Indeed, the
134·5 protein bears nuclear import and export signals that direct protein shuttling between the cytoplasm, nucleus and nucleolus (Cheng et al., 2002
). These observations are concordant with findings that the
134·5 protein of HSV-1(F) accumulates in both the nucleus and cytoplasm during virus infection (Ackermann et al., 1986
; Harland et al., 2003
). This dynamic process is thought to coordinate the different activities associated with the
134·5 protein during virus infection (Cheng et al., 2002
).
The precise role of the triplet repeats in HSV infection is not well understood. In the present study, the triplet repeats in the 134·5 protein encoded by HSV-1(F) in virus-infected cells were further examined. We show that deletions of the triplet repeats have no effect on the virus response to interferon involving PKR. When the number of triplet repeats is reduced to three or zero, virus egress is delayed or impaired compared with wild-type virus in mouse 3T6 cells. These results suggest that deletion of the triplet repeats in the
134·5 protein of HSV-1 modulates virus egress, but not virus response to interferon.
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METHODS |
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Plasmids.
Plasmid pRB143 contains the BamHI S fragment of HSV-1(F) in the BamHI site of pBR322. Construction of plasmids pJL0105 and pJL0206 was carried out by a two-step PCR using pRB143 as template (94 °C for 5 min and then 30 cycles of 94 °C, 30 s; 67 °C, 30 s; 72 °C, 2 min). To construct pJL0105, a PCR fragment was amplified with OligBH0018 (nt 488511 relative to the genome of HSV-1 17+) and OligJL0106 (nt 991961) and a second PCR fragment was amplified with OligJL0107 (nt 9791057) and OligBH0020 (nt 13311354). The resulting PCR products were used as templates and a final BstEIIDraIII fragment was amplified with OligBH0018 and OligBH0020 and ligated into the BstEII and DraIII sites of pRB143, yielding pJL0105. In this plasmid, the triplet repeats in the 134·5 gene were eliminated. The BamHI fragment from pJL0105 was then cloned into the BamHI site of pKO5, yielding pJL0106. To construct pJL0206, a PCR fragment was amplified with OligBH0018 and OligJL0207 (nt 9711043) and a second PCR fragment was amplified with OligJL0208 (nt 9911058) and OligBH0020. The resulting PCR products were used as templates and a final BstEIIDraIII fragment was amplified with OligBH0018 and OligBH0020 and ligated into the BstEII and DraIII sites of pRB143, yielding pJL0206. In this plasmid, the triplet repeats in the
134·5 gene were reduced from ten to three. The BamHI fragment from pJL0206 was then cloned into the BamHI site of pKO5Y, yielding pJL0207. To construct pJL0220, an EcoRIXhoI fragment was amplified by PCR using primers OligJL0211 (nt 490521) and OligJL0212 (nt 886919) and inserted into the EcoRI and XhoI sites of pGEX4T-1 (Pharmacia). In this plasmid, the region encoding aa 2146 of the
134·5 protein was fused in-frame to the glutathione S-transferase (GST) gene.
Antibodies.
The primary antibodies used include anti-134·5 antibody produced in this study, anti-HSV-1 antibody (Dako), anti-eIF2
antibody and anti-phospho-eIF2
antibody (Cell Signalling Technology and Biosource). Rabbit anti-
134·5 antibody was prepared as follows. Expression of the GST
134·5 fusion protein was induced by addition of 1 mM IPTG to the medium with Escherichia coli BL21 cells transformed with pJL0220, followed by affinity purification of the fusion protein from bacterial lysates on agarose beads conjugated with glutathione. The fusion protein was used for immunization of rabbits for production of polyclonal antibody.
Southern blot analysis.
Vero cells were infected with viruses at 10 p.f.u. per cell. At 18 h after infection, cells were harvested and resuspended in ice-cold TE buffer (pH 7·8) containing NP-40 (0·5 %) and RNase A (50 µg ml1). The cytoplasmic fraction was collected and treated with proteinase K (0·5 mg ml1) for 30 min at 37 °C. Viral DNAs were prepared and subjected to restriction digestions, electrophoretic separation in agarose gels, transfer to nitrocellulose membranes and hybridization with 32P-labelled DNA fragments as described previously (Chou & Roizman, 1994).
Virus growth assay.
Monolayers of mouse 10T1/2, CV-1 or 3T6 cells were infected with viruses either at 0·01 or 10 p.f.u. per cell. After adsorption for 2 h, the monolayers were overlaid with DMEM and incubated at 37 °C. At 24, 48 and 72 h post-infection, samples were harvested and viruses, released by three cycles of freezing and thawing, were titrated on Vero cells.
Immunoblotting.
Virus-infected cells were washed, harvested and solubilized in disruption buffer containing 50 mM Tris/HCl (pH 7·0), 5 % 2-mercaptoethanol, 2 % SDS and 2·75 % sucrose. Samples were then sonicated, boiled, subjected to electrophoresis on denaturing 12 % polyacrylamide gels, transferred to nitrocellulose membranes, blocked with 5 % non-fat milk and reacted with a selected primary antibody. The membranes were rinsed in PBS and reacted with donkey anti-rabbit immunoglobulin conjugated to horseradish peroxidase. Protein bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Interferon assay.
Monolayers of Vero cells grown to 80 % confluence were either untreated or pretreated with human leukocyte alpha interferon (IFN-) (1000 U ml1; Sigma) for 20 h. Cells were then infected with viruses at 0·05 p.f.u. per cell and incubated at 37 °C. At 48 h after infection, cells were harvested and virus yields were determined on Vero cells.
Electron microscopy analysis.
Monolayers of MEF 3T6 cells were infected with viruses at 0·5 p.f.u. per cell in 35 mm dishes. At 24 h post-infection, samples were first fixed in 4 % glutaraldehyde with 100 mM phosphate buffer (pH 6·87·2), fixed in 1 % osmium tetroxide in phosphate buffer, dehydrated in a series of ethanol concentrations (50, 70, 85, 95 and 100 % ethanol) and embedded in LX112 resin (Ladd Research Industries). Samples were removed from the Petri dishes and remounted on aluminium stubs. Ultrathin sections were cut with a Leica Utracut UCT, placed on 200-mesh copper grids and stained with uranyl acetate and lead citrate. Grids were viewed using a JEOL 1220 transmission electron microscope at 80 kV. Images were taken with a digital CCD camera (Software Digital Micrograph Gatan).
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RESULTS |
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DISCUSSION |
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Previous studies have demonstrated that the 134·5 protein inhibits the interferon response through the carboxyl- but not the amino-terminal domains (Cheng et al., 2001a
; Chou & Roizman, 1994
). The role of the triplet repeats has not been investigated. The data presented in this study show that, when the triplet repeats were reduced to three or zero, the mutant viruses were still resistant to interferon, like wild-type virus. These mutants replicated efficiently in CV-1 and mouse 10T1/2 cells, which are non-permissive to the
134·5 null mutants due to the PKR response. Furthermore, these triplet deletion mutants were capable of blocking the shutoff of protein synthesis and virus-induced eIF-2
phosphorylation was inhibited or reduced. As the
134·5 protein recruits PP1 to form a high-molecular-mass complex that dephosphorylates eIF-2
(Cerveny et al., 2003
; He et al., 1997b
), these results suggest that the triplet repeats are not essential for the functional interaction between the
134·5 protein, PP1 and eIF-2
in HSV-infected cells. This is consistent with the model that the carboxyl terminus of the
134·5 protein is a functional module that dictates virus response to interferon.
Although they replicated efficiently in CV-1 and 10T1/2 cells, the triplet deletion mutants grew with delayed kinetics in MEF 3T6 cells and did not replicate as well as wild-type virus. The different phenotypes associated with these mutants suggest that 3T6 cells either lack a cellular factor required for efficient virus egress or express an inhibitory factor that limits virus egress. Notably, these differences were most evident at 24 h post-infection. As virus release was reduced for these mutants, the triplet repeats appear to determine efficiency of virus egress in this cell line. Electron microscopic analysis revealed that, when infected with the triplet deletion mutants, significant numbers of virus particles were trapped in the perinuclear region or cytoplasm at 24 h after infection. Thus, HSV-1(F) with ten triplet repeats tends to be released more efficiently from infected cells than viruses with three or zero triplet repeats. In this context, it is notable that HSV-1 strains with a different number of the triplet repeats display different pathological properties in vivo (Bower et al., 1999; Mao & Rosenthal, 2003
; Perng et al., 2002
). It is speculated that these variations may have evolved to regulate virus egress and consequently the outcome of HSV infection.
The triplet repeats in the central domain are predicted to form secondary structures consisting of a collagen-like helix that bridges the amino- and carboxyl-terminal domains of the 134·5 protein. It is reasonable to predict that variation in the central domain modulates activity of the
134·5 protein. Recent experiments have indicated that the triplet repeats affect localization of the
134·5 protein in transfected cells (Mao & Rosenthal, 2002
). The
134·5 variants with 18 or more triplet repeats localize to the cytoplasm, whereas the ones with three triplet repeats localize to the nucleus. The
134·5 protein encoded by HSV-1(F) has ten triplet repeats and shuttles between the nucleolus, nucleus and cytoplasm (Cheng et al., 2002
). Indeed, the
134·5 protein bears nuclear import and export signals that dictate its cellular distribution (Cheng et al., 2002
). Given that the amino terminus of the
134·5 protein promotes nuclear as well as cytoplasmic egress (Jing et al., 2004
), it may represent a functional domain. Thus, one possibility is that the effect of the triplet repeats on virus egress may result from an altered cellular localization of the
134·5 protein. In this context, it is noted that, when transiently expressed in cells, the triplet deletion mutants are predominantly localized to the nucleus, whereas the wild-type
134·5 protein is localized to the cytoplasm and nucleus (data not shown). An alternative possibility is that the triplet repeats may modulate virus egress independently of the function required for shuttling of the
134·5 protein. Experiments are in progress to address these issues. It is notable that the
134·5 gene overlaps with ORF-P and ORF-O in the HSV-1 genome (Bohenzky et al., 1995
; Lagunoff & Roizman, 1994
). However, unlike
134·5, ORF-P and ORF-O are not expressed during lytic infection because ICP4 binds to a cis-element in the promoter of ORF-P/ORF-O (Lagunoff & Roizman, 1995
; Randall et al., 1997
). Thus, it is unlikely that the observed phenotype results from an alteration in ORF-P/ORF-O. Collectively, our results are concordant with previous findings that deletion of the
134·5 gene leads to defective virus egress and growth in infected cells (Brown et al., 1994b
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 March 2005;
accepted 20 May 2005.
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