Characterization of the triplet repeats in the central domain of the {gamma}134·5 protein of herpes simplex virus 1

Xianghong Jing and Bin He

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The {gamma}134·5 protein of herpes simplex virus 1 (HSV-1) consists of an amino-terminal domain, a central domain with triplet repeats (Ala–Thr–Pro) and a carboxyl-terminal domain. The triplet repeats are a unique feature of the {gamma}134·5 protein encoded by HSV-1, but the number of repeats varies among different strains. Notably, the central domain containing the triplet repeats is implicated in neuroinvasion. In this report, it has been shown that partial or full deletion of triplet repeats, i.e. from ten to either three or zero, in the {gamma}134·5 protein has no effect on the virus response to interferon. The triplet deletion mutants replicate efficiently in CV-1 and mouse 10T1/2 cells. However, in mouse 3T6 cells, these mutants grow with delayed growth kinetics. This decrease in growth, compared with wild-type HSV-1(F), does not result from failure of the virus to suppress the RNA-dependent protein kinase response, but rather from a delay in virus release or egress. Accordingly, these mutant viruses are predominantly present within infected cells. These results indicate that deletions in the central domain of the {gamma}134·5 protein impair virus egress, but not virus response to interferon.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The {gamma}134·5 protein of herpes simplex virus 1 (HSV-1; also known as Human herpesvirus 1) plays a pivotal role in virus virulence (Chou et al., 1990; MacLean et al., 1991; Whitley et al., 1993). Mutants that fail to express the {gamma}134·5 protein are incapable of multiplying in the brain and cannot cause encephalitis in experimental animal models (Chou et al., 1990; Whitley et al., 1993). In HSV-infected cells, the {gamma}134·5 protein inhibits the shutoff of protein synthesis mediated by double-stranded RNA-dependent protein kinase (PKR) (Chou & Roizman, 1992; He et al., 1997a). In this process, the {gamma}134·5 protein recruits protein phosphatase 1 (PP1), forming a high-molecular-mass complex that dephosphorylates eIF-2{alpha} and thereby prevents translation arrest (He et al., 1997b, 1998). This activity is linked to HSV resistance to interferon (Cheng et al., 2001a, b). Importantly, {gamma}134·5 null mutants display a virulent phenotype in PKR knockout mice, but not in wild-type mice (Chou et al., 1990; Leib et al., 2000). Studies show that the {gamma}134·5 protein is also involved in virus egress or release (Brown et al., 1994b). In mouse 3T6 cells, {gamma}134·5 mutants are defective in nuclear as well as cytoplasmic egress (Jing et al., 2004). Interestingly, the {gamma}134·5 protein blocks surface expression of MHC class II molecules in virus-infected cells, which is believed to impair the functions of CD4+ T cells (Trgovcich et al., 2002). Recently, it has been reported that the {gamma}134·5 protein interferes with autophagy (Tallóczy et al., 2002).

The {gamma}134·5 protein of HSV-1 consists of a large amino-terminal domain, a linker region of triplet repeats (Ala–Thr–Pro) 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 {gamma}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 {gamma}134·5 protein. However, the triplet repeats are not present in the {gamma}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 {gamma}134·5 protein in transfected mammalian cells (Mao & Rosenthal, 2002). Indeed, the {gamma}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 {gamma}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 {gamma}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 {gamma}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 {gamma}134·5 protein of HSV-1 modulates virus egress, but not virus response to interferon.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
Vero, 143tk, CV-1 and mouse embryo fibroblast (MEF) 3T6 cell lines were obtained from the ATCC and propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % (Vero) or 10 % (143tk, CV-1 and MEF 3T6) fetal bovine serum. HSV-1(F) is a prototype HSV-1 strain used in these studies (Ejercito et al., 1968). In recombinant virus R3616, a 1 kb fragment from the coding region of the {gamma}134·5 gene was deleted (Chou et al., 1990). To construct recombinant viruses JL0109R and JL0257R, plasmids pJL0106 and pJL0207 were transformed into an Escherichia coli RR1 strain that harboured wild-type HSV-BAC (bacterial artificial chromosome) as described previously (Cerveny et al., 2003). Positive clones were used to prepare HSV-BAC DNA with the Qiagen plasmid purification kit. Viral DNA was transfected into Vero cells using Lipofectamine reagent (Invitrogen). Virus was harvested 3–4 days after transfection and amplified on Vero cells. To restore the thymidine kinase (tk) gene, recombinant viral DNA was transfected along with plasmid pRB4867 containing the tk gene into Vero cells. The recombinant progeny was selected and purified on 143tk cells overlaid with HAT medium (0·1 mM sodium hypoxanthine, 0·4 µM aminopterin and 16 µM thymidine). Preparation of virus stock and titration of infectivity were carried out on Vero cells.

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 488–511 relative to the genome of HSV-1 17+) and OligJL0106 (nt 991–961) and a second PCR fragment was amplified with OligJL0107 (nt 979–1057) and OligBH0020 (nt 1331–1354). The resulting PCR products were used as templates and a final BstEII–DraIII 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 {gamma}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 971–1043) and a second PCR fragment was amplified with OligJL0208 (nt 991–1058) and OligBH0020. The resulting PCR products were used as templates and a final BstEII–DraIII 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 {gamma}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 EcoRI–XhoI fragment was amplified by PCR using primers OligJL0211 (nt 490–521) and OligJL0212 (nt 886–919) and inserted into the EcoRI and XhoI sites of pGEX4T-1 (Pharmacia). In this plasmid, the region encoding aa 2–146 of the {gamma}134·5 protein was fused in-frame to the glutathione S-transferase (GST) gene.

Antibodies.
The primary antibodies used include anti-{gamma}134·5 antibody produced in this study, anti-HSV-1 antibody (Dako), anti-eIF2{alpha} antibody and anti-phospho-eIF2{alpha} antibody (Cell Signalling Technology and Biosource). Rabbit anti-{gamma}134·5 antibody was prepared as follows. Expression of the GST–{gamma}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 ml–1). The cytoplasmic fraction was collected and treated with proteinase K (0·5 mg ml–1) 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-{alpha}) (1000 U ml–1; 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 6·8–7·2), 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).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of recombinant viruses with deletions in the triplet repeats (Ala–Thr–Pro) of the {gamma}134·5 protein
As an initial step, two deletion mutants were constructed. In JL0109R, there was a complete deletion of ten triplet repeats in the {gamma}134·5 protein. In JL0257R, only three triplet repeats were retained in the {gamma}134·5 protein (Fig. 1a). The objective was to assess how triplet repeats affect virus replication during HSV infection. Recombinant viruses were constructed using the BAC system as described in Methods. The BAC plasmid inserted in the tk gene was removed by co-transfection of viral DNA and a plasmid containing the tk gene into Vero cells. To verify the virus constructs, Southern blot analysis was carried out after BamHI, BstEII and DraIII digestion of viral DNAs (Fig. 1b). As expected, HSV-1(F), R3616, JL0109R and JL0257R yielded a 3 kb BamHI Q fragment containing the tk gene (Fig. 1b, upper panel, lanes 2–5). In addition, HSV-1(F) yielded a 526 bp BstEII–DraIII fragment representing the wild-type {gamma}134·5 gene. Due to deletions of the triplet repeats, JL0109R yielded a 436 bp BstEII–DraIII fragment and JL0257R yielded a 463 bp BstEII–DraIII fragment (Fig. 1b, lower panel, lanes 3 and 4, respectively). To examine protein expression, Western blot analysis was performed using anti-{gamma}134·5 antibody and anti-Us11 antibody. The results in Fig. 1(c) show that, in virus-infected cells, HSV-1(F), JL0109R and JL0257R expressed {gamma}134·5 proteins with different molecular masses (upper panel, lanes 2, 4 and 5), whereas the {gamma}134·5 protein was not detected in cells infected with R3616, which lacks the {gamma}134·5 gene (upper panel, lane 3). Notably, the {gamma}134·5 variants were produced in the same abundance as the wild-type {gamma}134·5 protein. Furthermore, comparable levels of Us11 were expressed in all virus-infected cells (Fig. 1c, lower panel, lanes 2–5).



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Fig. 1. (a) Schematic representation of the genome structure of HSV-1 and its derivatives. The two covalently linked components of HSV-1 DNA, L and S, each consist of unique sequences, UL and US, respectively, flanked by inverted repeats (Sheldrick & Berthelot, 1975). HSV-1(F) is the prototype strain used in this laboratory. In R3616, the coding region between the BstEII and StuI sites of the {gamma}134·5 gene is deleted. In recombinant virus JL0109R, the Ala–Thr–Pro repeats of the {gamma}134·5 gene are deleted. In recombinant virus JL0257R, only three Ala–Thr–Pro repeats are present in the {gamma}134·5 gene. Restriction site designations: N, NcoI; Be, BstEII. S, SacI; St, StuI. (b) Autoradiographic images of viral DNAs. Vero cells were infected with the indicated viruses at 10 p.f.u. per cell. At 18 h post-infection, cells were harvested and viral DNA was prepared and then digested with either BamHI or BstEII plus DraIII. Samples were electrophoretically separated on 0·8 % agarose gels and transferred to a nitrocellulose membrane. The tk gene was detected by hybridization to a 32P-labelled BamHI Q fragment of HSV-1. Similarly, the {gamma}134·5 gene was probed with a 32P-labelled {gamma}134·5 fragment spanning nt 859–1043. Plasmid pRB4867 contained the HSV-1(F) BamHI Q fragment encompassing the tk gene and plasmid pMC0001 contained a BamHI–SphI fragment encompassing the {gamma}134·5 gene. (c) Expression of the {gamma}134·5 protein. Vero cells were either mock-infected or infected with the indicated viruses at 10 p.f.u. per cell. At 18 h post-infection, cells were harvested and subjected to electrophoresis and transferred to nitrocellulose membranes, which were reacted with anti-{gamma}134·5 antibody and anti-Us11 antibody (a gift from R. Roller, University of Iowa, Iowa City, USA).

 
Deletions in the triplet repeats of the {gamma}134·5 gene do not affect virus response to IFN-{alpha}
To explore the role of the triplet repeats in virus response to interferon, virus growth was measured in Vero cells. In this experiment, monolayers of Vero cells were untreated or pretreated with IFN-{alpha} (1000 U ml–1) to induce the antiviral state. Cells were then infected with the indicated viruses and virus yields were determined 48 h after infection. As seen in Fig. 2, in the absence of interferon, HSV-1(F) and R3616 reached titres of 4·7x108 and 2·6x107 p.f.u. ml–1, respectively. Similarly, JL0109R and JL0257R replicated to titres of 2·0x108 and 4·9x107 p.f.u. ml–1, respectively. When cells were pretreated with interferon, replication of HSV-1(F) decreased slightly (4-fold), with a titre of 8·2x107 p.f.u. ml–1. Because of the deletion of the {gamma}134·5 gene, replication of R3616 decreased dramatically to a titre of 2·5x104 p.f.u. ml–1, exhibiting an interferon-sensitive phenotype. Under these conditions, JL0109R and JL0257R still replicated efficiently, reaching titres of 5·13x107 and 2·5x107 p.f.u. ml–1, respectively. Thus, like wild-type HSV-1(F), recombinant viruses with deletions in the triplet repeats are capable of blocking the antiviral action of IFN-{alpha}. It is concluded from this experiment that the triplet repeats in the {gamma}134·5 protein are not required to confer virus resistance to interferon.



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Fig. 2. Virus response to IFN-{alpha}. Monolayers of Vero cells were either untreated or pretreated with human leukocyte IFN-{alpha} (1000 U ml–1; 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 post-infection, cells were harvested and virus yields were determined on Vero cells. Data are means from three independent experiments, with standard deviation indicated. Open bars, not IFN-{alpha}-pretreated; shaded bars, IFN-{alpha}-pretreated.

 
Viruses with deletions in the triplet repeats of the {gamma}134·5 gene exhibit differential growth properties in mammalian cell lines
Virus growth properties were further evaluated in CV-1 cells and mouse 10T1/2 cells. These cell lines are restrictive to the {gamma}134·5 null mutant due to the shutoff of protein synthesis triggered by viral DNA synthesis. In this set of experiments, monolayers of cells were infected with viruses and virus yields were measured at different time points after infection. The results in Fig. 3(a) show that, in CV-1 cells, HSV-1(F) replicated to a titre of 2·0x106 p.f.u. ml–1 24 h after infection. This virus maintained efficient growth, reaching a titre of 2·9x107 p.f.u. ml–1 at 72 h after infection. As expected, R3616 replicated poorly, with titres of 1·32x103 and 2·6x104 p.f.u. ml–1 at 24 and 72 h, respectively. There was an approximately 1000-fold decrease in virus yield for R3616. Over the same growth period, JL0109R replicated as efficiently as wild-type HSV-1(F), with titres reaching 1·7x106 and 2·8x107 p.f.u. ml–1 at 24 and 72 h, respectively. JL0257R displayed a similar growth pattern to JL0109R. A virtually identical situation was also observed for these viruses in mouse 10T1/2 cells (Fig. 3b). Therefore, the growth properties of the recombinant viruses JL0109R and JL0257R are indistinguishable from those of wild-type HSV-1 (F) in these cell lines.



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Fig. 3. Growth properties of wild-type HSV-1(F) and the {gamma}134·5 mutants in CV-1 and mouse 10T1/2 cells. Confluent monolayers of CV-1 (a) or mouse 10T1/2 (b) cells were infected with HSV-1(F) ({blacklozenge}), R3616 ({blacksquare}), JL0109R ({blacktriangleup}) or JL0257R (x) at 0·01 p.f.u. per cell and incubated at 37 °C. Viruses were harvested at 24, 48 and 72 h post-infection. Samples were freeze–thawed three times and titrated on Vero cells at 37 °C. Data are means from three independent experiments; error bars indicate standard deviations.

 
Virus growth patterns were then assessed in MEF 3T6 cells, in which the {gamma}134·5 null mutants exhibit growth defects (Brown et al., 1994a, b). Monolayers of cells were infected with the indicated viruses and virus yields were then determined at 6, 12, 24, 48 and 72 h post-infection. Fig. 4 shows that wild-type HSV-1(F) replicated to a titre of 2·02x104 p.f.u. ml–1 at 12 h and grew to 1·23x107 p.f.u. ml–1 at 24 h post-infection. It continued to maintain efficient replication throughout infection, with a titre of 4·13x107 p.f.u. ml–1 at 72 h after infection. In contrast, R3616 replicated poorly, with a titre of 2·6x102 p.f.u. ml–1 at 24 h post-infection, which increased to a titre of 1·7x104 p.f.u. ml–1 at 72 h post-infection. Notably, JL0109R replicated to a titre of only 1·57x103 at 12 h. Although the titre of JL0109R increased to 3·74x105 p.f.u. ml–1 at 24 h, virus yield was still 33-fold less than that of HSV-1(F). Similarly, JL0257R replicated to a titre of 3·33x102 p.f.u. ml–1 at 12 h, with a titre of only 7·18x104 p.f.u. ml–1 at 24 h. There was 171-fold decrease in virus replication for JL0257R compared with the wild-type. Interestingly, this reduction was not fully restored until 72 h after infection. Hence, alterations in the triplet repeat in the {gamma}134·5 protein delayed virus replication in MEF 3T6 cells. These results indicated that reduction of the triplet repeats from ten to three or zero impairs virus replication in MEF 3T6 cells.



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Fig. 4. Growth properties of wild-type HSV-1(F) and the {gamma}134·5 mutants in MEF 3T6 cells. Confluent monolayers of MEF 3T6 cells were infected with HSV-1(F), R3616, JL0109R or JL0257R at 0·01 p.f.u. per cell and incubated at 37 °C. Viruses were harvested at 6, 12, 24, 48 and 72 h post-infection. Samples were freeze–thawed three times and titrated on Vero cells at 37 °C. Data are means from three independent experiments; error bars indicate standard deviations. For key, see legend to Fig. 3.

 
To address whether reduced virus replication in MEF 3T6 cells resulted from failure to counteract the PKR response, viral protein production was examined by Western blot analysis using anti-HSV antibodies. As indicated in Fig. 5(a), a high level of viral protein was detected in cells infected with HSV-1(F), JL0109R or JL0257R. In sharp contrast, little or no viral protein was detected in MEF 3T6 cells that were either mock-infected or infected with R3616. In parallel experiments, eIF-2{alpha} was analysed. Results in Fig. 5(b) show that eIF-2{alpha} was expressed in both mock-infected cells and cells infected with viruses. Phosphorylated eIF-2{alpha} was not detected in mock-infected cells or cells infected with HSV-1(F). However, a significant amount of phosphorylated eIF-2{alpha} was seen in cells infected with R3616 (Fig. 5b). In cells infected with the triplet deletion mutants, little or no phosphorylated eIF-2{alpha} was detected. The triplet deletion mutants were capable of preventing the translation shutoff mediated by PKR in 3T6 cells. Thus, the growth defect associated with JL0109R and JL0257R largely derived from a defect(s) after viral protein translation.



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Fig. 5. (a) Synthesis of viral proteins in virus-infected MEF 3T6 cells. Confluent monolayers of MEF 3T6 cells were either mock-infected or infected with the indicated viruses at 10 p.f.u. per cell. At 16 h post-infection, cells were harvested, solubilized, subjected to polyacrylamide gel analysis, transferred to a nitrocellulose sheet and reacted with polyclonal antibodies against whole HSV-1 antigens (Dako). (b) Phosphorylation state of eIF-2{alpha}. The same membrane from (a) was stripped and probed with antibodies against eIF-2{alpha} (upper panel) and phosphorylated eIF-2{alpha} (lower panel) (Cell Signal Technology and Biosource).

 
Virus release is decreased in MEF 3T6 cells infected with the triplet repeat deletion mutants
Based on the above analysis, levels of cell-associated and cell-free viruses were measured. Data in Table 1 show that, in HSV-1(F)-infected MEF 3T6 cells, total virus yield was 8·93x107 p.f.u. ml–1; 63 % of the virus was found in the cell body and 37 % was present in the medium. As expected, in cells infected with R3616, the overall virus yield was low, with a titre of 1·73x104 p.f.u. ml–1. There was an approximately 1000-fold drop in virus production compared with HSV-1(F). Moreover, a larger fraction (80 %) was associated with cells and a smaller fraction (20 %) was detected in the medium. Virus release into the medium was reduced by 17 % compared with HSV-1(F). Interestingly, in cells infected with JL0109R, the total virus yield was 8·77x106 p.f.u. ml–1, which was close to that for HSV-1(F). However, a large fraction of the virus (70 %) was associated with cells and a smaller portion (30 %) was released into the medium. There was a 7 % decrease in virus release compared with HSV-1(F). In addition, in cells infected with JL0257R, only 21·5 % of total virus was released into the medium, which is around a 15 % drop compared with HSV-1(F). Therefore, despite the quantitative difference, virus release was less efficient in cells infected with R3616, JL0109R and JL0257R. However, overall virus production for JL0109R and JL0257R was about 79- and 504-fold greater than that for R3616, respectively. This correlates with the ability of JL0109R and JL0257R to overcome the PKR response. Notably, these phenotypes were not apparent in CV-1 cells, where the relative distribution of cell-associated and cell-free viruses was similar for HSV-1(F), JL0109R and JL0257R.


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Table 1. Yields of secreted and cell-associated infectious virions

Confluent monolayers of MEF 3T6 and CV-1 cells were infected with HSV-1(F), R3616, JL0109R and JL0257R at 0·5 p.f.u. per cell and incubated at 37 °C. At 24 h post-infection, cell-associated viruses and viruses in the supernatant were collected separately and titrated on Vero cells. Data are means±SD (p.f.u. ml–1) from three independent experiments; percentages are given in parentheses.

 
The triplet repeats in the {gamma}134·5 protein are required for efficient virus egress in MEF 3T6 cells
Localization of viruses in MEF 3T6 cells was further analysed. Cells were infected with viruses and processed for electron microscopic analysis 24 h after infection. As expected, in cells infected with HSV-1(F), virus particles were evident not only in the cytoplasm, but also in the extracellular space. A large number of vesicles containing virus particles was observed in the cytoplasm (Fig. 6a). In cells infected with R3616, there was a drastic decrease in overall virus particle production. In addition, virus particles were seen predominantly in the nucleus and perinuclear region. Vesicles containing virions in the cytoplasm were also absent under this condition (Fig. 6b). In cells infected with JL0109R and JL0257R, significant numbers of virus capsids and particles were present in infected cells. However, distribution of the virus particles and cell nucleus morphology were similar to those observed in R3616-infected cells (Fig. 6c, d). Large numbers of virus particles were in areas close to or associated with the outer nuclear membrane. Relatively low levels were seen in the nucleus or on the cell surface. It seems that JL0109R and JL0257R are capable of budding into the cytoplasm from the nucleus, but are defective in reaching the cell surface.



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Fig. 6. Cellular distribution of viruses in MEF 3T6 cells. Confluent monolayers of MEF 3T6 cells were infected with HSV-1(F) (a), R3616 (b), JL0109R (c) or JL0257R (d) at 0·5 p.f.u. per cell. At 24 h post-infection, cells were first fixed in 4 % glutaraldehyde in 100 mM phosphate buffer (pH 6·8–7·2) and then fixed in 1 % osmium tetroxide. Cells were dehydrated in ethanol, embedded in LX112 resin and stained with uranyl acetate and lead citrate. Thin sections were prepared and viewed using a JEOL 1220 transmission electron microscope at 80 kV. Images were captured with a Gatan digital CCD camera. Nuc, Nuclear region; Cyt, cytoplasm. Arrowheads indicate virus particles. Bars, 1 µm.

 
To quantify the observed differences, subcellular distribution of virions was counted in 15–20 cells for each virus. As summarized in Table 2, in the absence of the {gamma}134·5 gene, a large fraction (30 %) of virus particles was trapped in the nucleus and 61 % were in the area around the nuclear membrane. In cells infected with JL0109R, around 73 % of virus particles accumulated in the outer nuclear membrane region, whereas 14 % were on the cell surface. Furthermore, in cells infected with JL0257R, roughly only 5·5 % of virus particles were on the cell surface and 88 % accumulated in the outer nuclear membrane region. JL0257R was more defective in cytoplasmic egress than JL0109R. These results correlate with a delay in virus growth kinetics and a decrease in release. Collectively, these data suggest that the triplet repeats of the {gamma}134·5 protein play an important role in facilitating virus cytoplasmic egress.


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Table 2. Distribution of virions in MEF 3T6 cells

Virus particles present in the nucleus, perinuclear region and cytoplasm and on the cell surface were counted in electron micrographs of at least 15–20 randomly sampled MEF 3T6 cells infected with the indicated viruses. The mean numbers of virus particles per cellular compartment are shown and the numbers in parentheses denote percentages of virus particles in the different sections of a cell.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Several lines of evidence have indicated that the {gamma}134·5 protein is a multifunctional protein, which consists of an amino-terminal domain, a central domain with triplet repeats (Ala–Thr–Pro) and a carboxyl-terminal domain (Bower et al., 1997, 1999; Cheng et al., 2003; Chou & Roizman, 1990, 1992; Jing et al., 2004; Tallóczy et al., 2002; Trgovcich et al., 2002). The carboxyl-terminal domain acts to prevent the interferon response, involving PKR (Cheng et al., 2001a; Chou & Roizman, 1994). It has recently been found that, in MEF 3T6 cells, the amino-terminal domain is required for virus egress (Jing et al., 2004). The central domain in the {gamma}134·5 protein encoded by HSV-1 is implicated in neuroinvasion (Bower et al., 1999; Mao & Rosenthal, 2003; Perng et al., 2002). To extend these studies, the triplet repeats in the central domain of the {gamma}134·5 protein encoded by HSV-1(F) were further examined. These data demonstrate that the triplet repeats in the central domain of the {gamma}134·5 protein facilitate virus egress but not interferon response in infected cells.

Previous studies have demonstrated that the {gamma}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 {gamma}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{alpha} phosphorylation was inhibited or reduced. As the {gamma}134·5 protein recruits PP1 to form a high-molecular-mass complex that dephosphorylates eIF-2{alpha} (Cerveny et al., 2003; He et al., 1997b), these results suggest that the triplet repeats are not essential for the functional interaction between the {gamma}134·5 protein, PP1 and eIF-2{alpha} in HSV-infected cells. This is consistent with the model that the carboxyl terminus of the {gamma}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 {gamma}134·5 protein. It is reasonable to predict that variation in the central domain modulates activity of the {gamma}134·5 protein. Recent experiments have indicated that the triplet repeats affect localization of the {gamma}134·5 protein in transfected cells (Mao & Rosenthal, 2002). The {gamma}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 {gamma}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 {gamma}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 {gamma}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 {gamma}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 {gamma}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 {gamma}134·5 protein. Experiments are in progress to address these issues. It is notable that the {gamma}134·5 gene overlaps with ORF-P and ORF-O in the HSV-1 genome (Bohenzky et al., 1995; Lagunoff & Roizman, 1994). However, unlike {gamma}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 {gamma}134·5 gene leads to defective virus egress and growth in infected cells (Brown et al., 1994b).


   ACKNOWLEDGEMENTS
 
We thank Bernard Roizman and Richard Roller for reagents. This work was supported by grant AI 46665 (B. H.) from the National Institute of Allergy and Infectious Diseases.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ackermann, M., Chou, J., Sarmiento, M., Lerner, R. A. & Roizman, B. (1986). Identification by antibody to a synthetic peptide of a protein specified by a diploid gene located in the terminal repeats of the L component of herpes simplex virus genome. J Virol 58, 843–850.[Medline]

Bohenzky, R. A., Lagunoff, M., Roizman, B., Wagner, E. K. & Silverstein, S. (1995). Two overlapping transcription units which extend across the L-S junction of herpes simplex virus type 1. J Virol 69, 2889–2897.[Abstract]

Bower, J. R., Mao, H., Durishin, C., Rozenbom, E., Detwiler, M., Rempinski, D., Karban, T. L. & Rosenthal, K. S. (1999). Intrastrain variants of herpes simplex virus type 1 isolated from a neonate with fatal disseminated infection differ in the ICP34·5 gene, glycoprotein processing, and neuroinvasiveness. J Virol 73, 3843–3853.[Abstract/Free Full Text]

Brown, S. M., Harland, J., MacLean, A. R., Podlech, J. & Clements, J. B. (1994a). Cell type and cell state determine differential in vitro growth of non-neurovirulent ICP34·5-negative herpes simplex virus types 1 and 2. J Gen Virol 75, 2367–2377.[Abstract]

Brown, S. M., MacLean, A. R., Aitken, J. D. & Harland, J. (1994b). ICP34·5 influences herpes simplex virus type 1 maturation and egress from infected cells in vitro. J Gen Virol 75, 3679–3686.[Abstract]

Brown, S. M., MacLean, A. R., McKie, E. A. & Harland, J. (1997). The herpes simplex virus virulence factor ICP34·5 and the cellular protein MyD116 complex with proliferating cell nuclear antigen through the 63-amino-acid domain conserved in ICP34·5, MyD116, and GADD34. J Virol 71, 9442–9449.[Abstract]

Cerveny, M., Hessefort, S., Yang, K., Cheng, G., Gross, M. & He, B. (2003). Amino acid substitutions in the effector domain of the {gamma}134·5 protein of herpes simplex virus 1 have differential effects on viral response to interferon-alpha. Virology 307, 290–300.[CrossRef][Medline]

Cheng, G., Brett, M. E. & He, B. (2001a). Val193 and Phe195 of the {gamma}134·5 protein of herpes simplex virus 1 are required for viral resistance to interferon-{alpha}/{beta}. Virology 290, 115–120.[CrossRef][Medline]

Cheng, G., Gross, M., Brett, M. E. & He, B. (2001b). AlaArg motif in the carboxyl terminus of the {gamma}134·5 protein of herpes simplex virus type 1 is required for the formation of a high-molecular-weight complex that dephosphorylates eIF-2{alpha}. J Virol 75, 3666–3674.[Abstract/Free Full Text]

Cheng, G., Brett, M. E. & He, B. (2002). Signals that dictate nuclear, nucleolar, and cytoplasmic shuttling of the {gamma}134·5 protein of herpes simplex virus type 1. J Virol 76, 9434–9445.[Abstract/Free Full Text]

Cheng, G., Yang, K. & He, B. (2003). Dephosphorylation of eIF-2{alpha} mediated by the {gamma}134·5 protein of herpes simplex virus type 1 is required for viral response to interferon but is not sufficient for efficient viral replication. J Virol 77, 10154–10161.[Abstract/Free Full Text]

Chou, J. & Roizman, B. (1990). The herpes simplex virus 1 gene for ICP34·5, which maps in inverted repeats, is conserved in several limited-passage isolates but not in strain 17syn+. J Virol 64, 1014–1020.[Medline]

Chou, J. & Roizman, B. (1992). The {gamma}134·5 gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programmed cell death in neuronal cells. Proc Natl Acad Sci U S A 89, 3266–3270.[Abstract/Free Full Text]

Chou, J. & Roizman, B. (1994). Herpes simplex virus 1 {gamma}134·5 gene function, which blocks the host response to infection, maps in the homologous domain of the genes expressed during growth arrest and DNA damage. Proc Natl Acad Sci U S A 91, 5247–5251.[Abstract/Free Full Text]

Chou, J., Kern, E. R., Whitley, R. J. & Roizman, B. (1990). Mapping of herpes simplex virus-1 neurovirulence to {gamma}134·5, a gene nonessential for growth in culture. Science 250, 1262–1266.[Medline]

Ejercito, P. M., Kieff, E. D. & Roizman, B. (1968). Characterization of herpes simplex virus strains differing in their effects on social behaviour of infected cells. J Gen Virol 2, 357–364.[Medline]

Harland, J., Dunn, P., Cameron, E., Conner, J. & Brown, S. M. (2003). The herpes simplex virus (HSV) protein ICP34·5 is a virion component that forms a DNA-binding complex with proliferating cell nuclear antigen and HSV replication proteins. J Neurovirol 9, 477–488.[Medline]

He, B., Chou, J., Liebermann, D. A., Hoffman, B. & Roizman, B. (1996). The carboxyl terminus of the murine MyD116 gene substitutes for the corresponding domain of the {gamma}134·5 gene of herpes simplex virus to preclude the premature shutoff of total protein synthesis in infected human cells. J Virol 70, 84–90.[Abstract]

He, B., Chou, J., Brandimarti, R., Mohr, I., Gluzman, Y. & Roizman, B. (1997a). Suppression of the phenotype of {gamma}134·5-herpes simplex virus 1: failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the {alpha}47 gene. J Virol 71, 6049–6054.[Abstract]

He, B., Gross, M. & Roizman, B. (1997b). The {gamma}134·5 protein of herpes simplex virus 1 complexes with protein phosphatase 1{alpha} to dephosphorylate the {alpha} subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A 94, 843–848.[Abstract/Free Full Text]

He, B., Gross, M. & Roizman, B. (1998). The {gamma}134·5 protein of herpes simplex virus 1 has the structural and functional attributes of a protein phosphatase 1 regulatory subunit and is present in a high molecular weight complex with the enzyme in infected cells. J Biol Chem 273, 20737–20743.[Abstract/Free Full Text]

Hollander, M. C., Zhan, Q., Bae, I. & Fornace, A. J., Jr (1997). Mammalian GADD34, an apoptosis- and DNA damage-inducible gene. J Biol Chem 272, 13731–13737.[Abstract/Free Full Text]

Jing, X., Cerveny, M., Yang, K. & He, B. (2004). Replication of herpes simplex virus 1 depends on the {gamma}134·5 functions that facilitate virus response to interferon and egress in the different stages of productive infection. J Virol 78, 7653–7666.[Abstract/Free Full Text]

Lagunoff, M. & Roizman, B. (1994). Expression of a herpes simplex virus 1 open reading frame antisense to the {gamma}134·5 gene and transcribed by an RNA 3' coterminal with the unspliced latency-associated transcript. J Virol 68, 6021–6028.[Abstract]

Lagunoff, M. & Roizman, B. (1995). The regulation of synthesis and properties of the protein product of open reading frame P of the herpes simplex virus 1 genome. J Virol 69, 3615–3623.[Abstract]

Leib, D. A., Machalek, M. A., Williams, B. R., Silverman, R. H. & Virgin, H. W. (2000). Specific phenotypic restoration of an attenuated virus by knockout of a host resistance gene. Proc Natl Acad Sci U S A 97, 6097–6101.[Abstract/Free Full Text]

Lord, K. A., Hoffman-Liebermann, B. & Liebermann, D. A. (1990). Sequence of MyD116 cDNA: a novel myeloid differentiation primary response gene induced by IL6. Nucleic Acids Res 18, 2823.[Medline]

MacLean, A. R., ul-Fareed, M., Robertson, L., Harland, J. & Brown, S. M. (1991). Herpes simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence-related sequences in Glasgow strain 17+ between immediate early gene 1 and the ‘a’ sequence. J Gen Virol 72, 631–639.[Abstract]

Mao, H. & Rosenthal, K. S. (2002). An N-terminal arginine-rich cluster and a proline-alanine-threonine repeat region determine the cellular localization of the herpes simplex virus type 1 ICP34·5 protein and Its ligand, protein phosphatase 1. J Biol Chem 277, 11423–11431.[Abstract/Free Full Text]

Mao, H. & Rosenthal, K. S. (2003). Strain-dependent structural variants of herpes simplex virus type 1 ICP34·5 determine viral plaque size, efficiency of glycoprotein processing, and viral release and neuroinvasive disease potential. J Virol 77, 3409–3417.[Abstract/Free Full Text]

McGeoch, D. J., Cunningham, C., McIntyre, G. & Dolan, A. (1991). Comparative sequence analysis of the long repeat regions and adjoining parts of the long unique regions in the genomes of herpes simplex viruses types 1 and 2. J Gen Virol 72, 3057–3075.[Abstract]

Perng, G. C., Mott, K. R., Osorio, N., Yukht, A., Salina, S., Nguyen, Q. H., Nesburn, A. B. & Wechsler, S. L. (2002). Herpes simplex virus type 1 mutants containing the KOS strain ICP34·5 gene in place of the McKrae ICP34·5 gene have McKrae-like spontaneous reactivation but non-McKrae-like virulence. J Gen Virol 83, 2933–2942.[Abstract/Free Full Text]

Randall, G., Lagunoff, M. & Roizman, B. (1997). The product of ORF O located within the domain of herpes simplex virus 1 genome transcribed during latent infection binds to and inhibits in vitro binding of infected cell protein 4 to its cognate DNA site. Proc Natl Acad Sci U S A 94, 10379–10384.[Abstract/Free Full Text]

Sheldrick, P. & Berthelot, N. (1975). Inverted repetitions in the chromosome of herpes simplex virus. Cold Spring Harb Symp Quant Biol 39, 667–678.[Medline]

Tallóczy, Z., Jiang, W., Virgin, H. W., IV, Leib, D. A., Scheuner, D., Kaufman, R. J., Eskelinen, E.-L. & Levine, B. (2002). Regulation of starvation- and virus-induced autophagy by the eIF2{alpha} kinase signaling pathway. Proc Natl Acad Sci U S A 99, 190–195.[Abstract/Free Full Text]

Trgovcich, J., Johnson, D. & Roizman, B. (2002). Cell surface major histocompatibility complex class II proteins are regulated by the products of the {gamma}134·5 and U(L)41 genes of herpes simplex virus 1. J Virol 76, 6974–6986.[Abstract/Free Full Text]

Whitley, R. J., Kern, E. R., Chatterjee, S., Chou, J. & Roizman, B. (1993). Replication, establishment of latency, and induced reactivation of herpes simplex virus {gamma}134·5 deletion mutants in rodent models. J Clin Invest 91, 2837–2843.[Medline]

Zhan, Q., Lord, K. A., Alamo, I., Jr & 7 other authors (1994). The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth. Mol Cell Biol 14, 2361–2371.[Abstract]

Received 14 March 2005; accepted 20 May 2005.



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