Functional interaction between the herpes simplex-1 DNA polymerase and UL42 protein

The herpes simplex virus 1 (HSV-1) UL42 protein, one of seven herpes-encoded polypeptides that are required for the replication of the HSV-1 genome, is found in a 1:1 complex with the HSV-1 DNA polymerase (Crute, J. J., and Lehman, I. R. (1989) J. Biol. Chem. 264, 19266-19270). To obtain herpes DNA polymerase free of UL42 protein, we have cloned and overexpressed the Pol gene in a recombinant baculovirus vector and purified the recombinant DNA polymerase to near homogeneity. Replication of singly primed M13mp18 single-stranded DNA by the recombinant enzyme in the presence of the herpes encoded single-stranded DNA-binding protein ICP8 yields in addition to some full-length product a distribution of intermediate length products by a quasi-processive mode of deoxynucleotide polymerization. Addition of the purified UL42 protein results in completely processive polymerization and the generation of full-length products. Similar processivity is observed with the HSV-1 DNA polymerase purified from herpes-infected Vero cells. Processive DNA replication by the DNA polymerase isolated from HSV-1-infected Vero cells or the recombinant DNA polymerase-UL42 protein complex requires that the single-stranded DNA be coated with saturating levels of ICP8. ICP8 which binds single-stranded DNA in a highly cooperative manner is presumably required to melt out regions of secondary structure in the single-stranded DNA template, thereby potentiating the processivity enhancing action of the UL42 protein.     

Autoproteolysis of herpes simplex virus type 1 protease releases an active catalytic domain found in intermediate capsid particles.

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.     

p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress

As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.     

Characterization of herpes simplex virus 1 alpha proteins 0, 4, and 27 with monoclonal antibodies.

Analyses of the reactivity and patterns of synthesis of infected cell polypeptides (ICPs) specified by herpes simplex virus (HSV) 1 and 2 and by HSV-1 X HSV-2 recombinants indicated that monoclonal antibody H1183 reacted with HSV-1 alpha ICP0, whereas monoclonal antibody H1113 reacted with both HSV-1 and HSV-2 alpha ICP27. H1083 and H1113 and a monoclonal antibody to ICP4 (H640) similar to one previously described (D. K. Braun et al., J. Virol. 46:103-112.) were then used to study the properties of these alpha proteins. The results were as follows: alpha ICP0, ICP4, and ICP27 accumulated primarily in the nuclei of infected cells. ICP4 and ICP27 were poorly soluble in nondenaturing buffer solutions. ICP0 was considerably more soluble than ICP4 and ICP27. ICP0, ICP4, and ICP27 were readily partially purified by immunoaffinity chromatography from lysates of infected cells solubilized with denaturing agents such as sodium dodecyl sulfate. ICP0 and ICP27 were phosphorylated in cells overlaid with medium containing 32P early (1 to 3 h) or late (18 to 20 h) postinfection. A fraction, but not all, 32P that was incorporated early was chased in the presence of unlabeled phosphate. ICP0, ICP4, and ICP27 labeled with either 32P or [35S]methionine yielded multiple spots upon two-dimensional separations. However, ICP4 quantitatively precipitated at the origin when the migration in the first dimension was from acid to base, and both ICP4 and ICP27 partially precipitated at the origin when the direction of migration was reversed.     

Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4

Using Vero cells transformed with the wild-type gene for ICP4 as the permissive host cell, we isolated herpes simplex virus type 1 (HSV-1) mutants containing deletions in both copies of the ICP4 gene. The mutants, d120 and d202, contained deletions of 4.1 and 0.5 kilobases, respectively, in each copy of ICP4. ICP4 mRNA synthesized in d202-infected Vero cells was 0.5 kilobases smaller than that synthesized in cells infected with the wild-type virus. No ICP4 mRNA was detected in d120-infected Vero cells. d120 and d202 specified polypeptides that reacted with ICP4 antiserum and were smaller than the wild-type ICP4 polypeptide by 130 and 30 kilodaltons, respectively. The only other HSV-1 gene products detectable on infection of Vero cells with d120 and d202 were ICP6 (of the early kinetic class of HSV-1 polypeptides), ICP0 (immediate early), ICP22 (immediate early), and ICP27 (immediate early). Immediate-early polypeptides ICP0 and ICP27 were expressed to a higher level in Vero cells infected with an ICP4 temperature-sensitive (ts) mutant (tsB32) at 39 degrees C, indicating immediate-early stimulatory activity associated with the ts ICP4 polypeptide. In addition, the patterns of complementation of d120, d202, and tsB32 in ICP4-transformed cells also demonstrated inhibitory activity associated with the ts form of the ICP4 polypeptide.     

Cells expressing herpes simplex virus glycoprotein gC but not gB, gD, or gE are recognized by murine virus-specific cytotoxic T lymphocytes

To determine which viral molecule(s) is recognized by herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL), target cells were constructed which express individual HSV glycoproteins. A mouse L cell line, Z4/6, which constitutively expressed high levels of HSV type 2 (HSV-2) gD (gD-2) was isolated and characterized previously (D. C. Johnson and J. R. Smiley, J. Virol. 54:682-689, 1985). Despite the expression of gD on the surface of Z4/6 cells, these cells were not killed by anti-HSV-2 CTL generated following intravaginal infection of syngeneic mice. In contrast, parental Z4 or Z4/6 cells infected with HSV-2 were lysed. Furthermore, unlabeled Z4/6 cells were unable to block the lysis of HSV-2-infected labeled target cells. Cells which express HSV-1 gB (gB-1) were isolated by transfecting L cells with the recombinant plasmid pSV2gBneo, which contains the HSV-1 gB structural sequences and the neomycin resistance gene coupled to the simian virus 40 early promoter and selecting G418-resistant cell lines. One such cell line, Lta/gB15, expressed gB which was detected by immunoprecipitation and at the cell surface by immunofluorescence. Additionally, cells expressing HSV-1 gC (gC-1) or gE (gE-1) were isolated by transfecting Z4 cells, which are L cells expressing ICP4 and ICP47, with either the recombinant plasmid pGE15neo, which contains the gE structural sequences and the neomycin resistance gene, or pDC17, which contains the gC structural gene coupled to the gD-1 promoter. A number of G418-resistant cell lines were isolated which expressed gC-1 or gE-1 at the cell surface. Anti-HSV-1 CTL generated following footpad infection of syngeneic mice were unable to lyse target cells expressing gB-1 or gE-1. In contrast, target cells expressing very low levels of gC-1 were killed as well as HSV-1-infected target cells. Furthermore, infection of gC-1-transformed target cells with wild-type HSV-1 or a strain of HSV-1 that does not express gC did not result in a marked increase in susceptibility to lysis. These results suggest that murine class I major histocompatibility complex-restricted anti-HSV CTL recognize gC-1 but do not recognize gB, gD, or gE as these molecules are expressed in transfected syngeneic target cells. The results are discussed in terms of recent evidence concerning the specificity of antiviral CTL.     

The herpes simplex virus UL37 protein is phosphorylated in infected cells.

The herpes simplex virus type 1 (HSV-1) UL37 open reading frame encodes a 120-kDa late (gamma 1), nonstructural protein in infected cells. Recent studies in our laboratory have demonstrated that the UL37 protein interacts in the cytoplasm of infected cells with ICP8, the major HSV-1 DNA-binding protein. As a result of this interaction, the UL37 protein is transported to the nucleus and can be coeluted with ICP8 from single-stranded DNA columns. Pulse-labeling and pulse-chase studies of HSV-1-infected cells with [35S]methionine and 32Pi demonstrated that UL37 was a phosphoprotein which did not have a detectable rate of turnover. The protein was phosphorylated soon after translation and remained phosphorylated throughout the viral replicative cycle. UL37 protein expressed from a vaccinia virus recombinant was also phosphorylated during infection, suggesting that the UL37 protein was phosphorylated by a cellular kinase and that interaction with the ICP8 protein was not a prerequisite for UL37 phosphorylation.     

Analysis of the role of autophagy in replication of herpes simplex virus in cell culture

The herpes simplex virus type 1 (HSV-1) neurovirulence gene encoding ICP34.5 controls the autophagy pathway. HSV-1 strains lacking ICP34.5 are attenuated in growth and pathogenesis in animal models and in primary cultured cells. While this growth defect has been attributed to the inability of an ICP34.5-null virus to counteract the induction of translational arrest through the PKR antiviral pathway, the role of autophagy in the regulation of HSV-1 replication is unknown. Here we show that HSV-1 infection induces autophagy in primary murine embryonic fibroblasts and that autophagosome formation is increased to a greater extent following infection with an ICP34.5-deficient virus. Elimination of the autophagic pathway did not significantly alter the replication of wild-type HSV-1 or ICP34.5 mutants. The phosphorylation state of eIF2alpha and viral protein accumulation were unchanged in HSV-1-infected cells unable to undergo autophagy. These data show that while ICP34.5 regulates autophagy, it is the prevention of translational arrest by ICP34.5 rather than its control of autophagy that is the pivotal determinant of efficient HSV-1 replication in primary cell culture.     

A 165 kd protein of the herpes simplex virion shares a common epitope with the regulatory protein, ICP4

We have investigated the possibility that immediate-early (IE) protein ICP4 could be a part of herpes simplex virus type 1 (HSV-1) virion particle. Immunodetection with a monoclonal antibody against ICP4 reveals that a component of the virion, migrating at 165 kd, shares a common epitope with this immediate-early protein. Immunolocalization studies on purified virions indicate that the antigen can be detected only in virions without membranes, and is located outside the capsid, most probably in the tegument. Ultrastructural localizations on HSV-1 infected BHK cells extracted with a nonionic detergent confirm that the protein immunoreacting with anti-ICP4 is present in virions.     

Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The large subunit of the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR1) is demonstrated to possess serine/threonine-specific kinase activity. Computer-assisted sequence analysis identified regions within the amino terminus of ICP10 that are homologous to the catalytic domain of known protein kinases (PKs). An in vitro kinase assay confirmed the ability of ICP10, immunoprecipitated from either HSV-2-infected cells or from cells transfected with an ICP10 expression vector, to autophosphorylate and transphosphorylate exogenous substrates in the presence of ATP and Mg2+. The HSV-1 RR1 was shown to be negative for PK activity under these conditions. PK activity was localized to a 57-kDa amino-terminal region within ICP10 that lacked RR activity.     

Glycoprotein gE of herpes simplex virus type 1: effects of anti-gE on virion infectivity and on virus-induced fc-binding receptors.

An Fc-binding glycoprotein, designated gE, was detected previously in cells infected with herpes simplex virus type 1 (HSV-1) and in virion preparations isolated from infected cells. For the studies reported here, we purified gE from HSV-1 strain HFEM(syn) by affinity chromatography and preparative electrophoresis and then immunized a rabbit to produce an antiserum to glycoprotein gE. We found that this antiserum selectively precipitated gE and its precursors from detergent-solubilized extracts of HSV-1 strain HFEM(syn)-infected HEp-2 cells, from extracts of other cell lines infected with the same virus, and from extracts of HEp-2 cells infected with several other HSV-1 strains. The antiserum did not precipitate any proteins from uninfected cells. The several forms of gE detected by immunoprecipitation accumulated in variable quantities in different cells infected with the different virus strains and also varied slightly with respect to electrophoretic mobility, suggesting some differences in the gE's from different HSV-1 strains and some effects of the host cell on the nature and extent of post-translational processing. One of the electrophoretic forms of gE previously detected in purified preparations of virions could be precipitated by anti-gE from extracts of purified HSV-1 strain HFEM(syn) virions. Moreover, anti-gE neutralized HSV-1 infectivity, but only in the presence of complement. Finally, F(ab')2 fragments of the anti-gE immunoglobulin partially inhibited the binding of 125I-labeled immunoglobulin G to the Fc receptors on HSV-1-infected cells.     

The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Is Required for Immediate-Early Gene Expression and Virus Growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10ΔPK). ICP10ΔPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10ΔPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10ΔPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.     

Herpes Simplex Virus Type 1 ICP27 Protein： Its Expression, Purification and Specific Antiserum Production

Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. Coli and purified by nickel-nitrilotriacetic acid (Ni2+-NTA) affinity resin column,finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.     

The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) blocks apoptosis in hippocampal neurons, involving activation of the MEK/MAPK survival pathway.

Herpes simplex virus type 1 (HSV-1) and HSV-2 trigger or counteract apoptosis by a cell-specific mechanism. Our studies are based on previous findings that the protein kinase (PK) domain of the large subunit of HSV-2 ribonucleotide reductase (ICP10) activates the Ras/MEK/MAPK pathway (Smith et al., J. Virol. 74:10417, 2000). Because survival pathways can modulate apoptosis, we used cells that are stably or transiently transfected with ICP10 PK, an HSV-2 mutant deleted in ICP10 PK (ICP10DeltaPK) and the MEK-specific inhibitor U0126 to examine the role of ICP10 PK in apoptosis. Apoptosis was induced by staurosporine or D-mannitol in human (HEK293) cells or HEK293 cells stably transfected with the ICP10 PK-negative mutant p139 (JHL15), as determined by morphology, DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage. HEK293 cells stably transfected with ICP10 (JHLa1) were protected from apoptosis. ICP10 but not p139 protected neuronally differentiated PC12 cells from death due to nerve growth factor withdrawal, and apoptosis (determined by TUNEL) and caspase-3 activation were seen in primary hippocampal cultures infected with ICP10DeltaPK but not with HSV-2 or a revertant virus [HSV-2(R)]. The data indicate that ICP10 has antiapoptotic activity under both paradigms and that it requires a functional PK activity. The apoptotic cells in primary hippocampal cultures were neurons, as determined by double immunofluorescence with fluorescein-labeled dUTP (TUNEL) and phycoerythrin-labeled antibodies specific for neuronal proteins (TuJ1 and NF-160). Protection from apoptosis was associated with MEK/MAPK activation, as evidenced by (i) increased levels of activated (phosphorylated) MAPK in HSV-2- but not ICP10DeltaPK-infected cultures and (ii) inhibition of MAPK activation by the MEK-specific inhibitor U0126. MEK and MAPK were activated by infection with UV-inactivated but not antibody-neutralized HSV-2, suggesting that activation requires cellular penetration but is independent of de novo viral protein synthesis.     

Interaction of herpes simplex virus 1 alpha regulatory protein ICP0 with elongation factor 1delta: ICP0 affects translational machinery.

The herpes simplex virus 1 (HSV-1)-infected cell protein 0 (ICP0) is a promiscuous transactivator, and by necessity, its functions must be mediated through cellular gene products. In an attempt to identify cellular factors interacting with ICP0, we used the carboxyl-terminal domain of ICP0 as "bait" in the yeast (Saccharomyces cerevisiae) two-hybrid system. Our results were as follows: (i) All 43 cDNAs in positive yeast colonies were found to encode the same translation factor, elongation factor delta-1 (EF-1delta). (ii) Purified chimeric protein consisting of glutathione S-transferase (GST) fused to EF-1delta specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate. (iii) Fractionation of infected HEp-2 cells and immunofluorescence studies revealed that ICP0 was localized both in the nucleus and in the cytoplasm. In primary human foreskin fibroblasts, ICP0 was localized predominantly in the cytoplasm throughout HSV-1 infection even early in infection. (iv) Addition of the chimeric protein GST-carboxyl-terminal domain of ICP0 to the rabbit reticulocyte lysate in vitro translation system resulted in a dose-dependent decrease in protein synthesis. In contrast, GST alone or GST fused to the amino-terminal domain of ICP0 had no effect on the in vitro translation system. (v) The predominant forms of EF-1delta on electrophoresis in denaturing gels have apparent Mrs of 38,000 and 40,000. The higher-Mr form is a minor species in mock-infected cells, whereas in human fibroblasts and Vero cells infected with HSV-1, this isoform becomes dominant. These results indicate that ICP0 is present and may have a significant role in the cytoplasm of infected cells, possibly by altering the efficiency of translation of viral mRNAs.     

CK2 protein kinase is stimulated and redistributed by functional herpes simplex virus ICP27 protein

It has been shown previously (S. Wadd, H. Bryant, O. Filhol, J. E. Scott, T.-T. Hsieh, R. D. Everett, and J. B. Clements, J. Biol. Chem. 274:28991-28998, 2000) that ICP27, an essential and multifunctional herpes simplex virus type 1 (HSV-1) protein, interacts with CK2 and with heterogeneous ribonucleoprotein K (hnRNP K). CK2 is a pleiotropic and ubiquitous protein kinase, and the tetrameric holoenzyme consists of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We show here that HSV-1 infection stimulates CK2 activity. CK2 stimulation occurs at early times after infection and correlates with redistribution of the holoenzyme from the nucleus to the cytoplasm. Both CK2 stimulation and redistribution require expression and cytoplasmic accumulation of ICP27. In HSV-1-infected cells, CK2 phosphorylates ICP27 and affects its cytoplasmic accumulation while it also phosphorylates hnRNP K, which is not ordinarily phosphorylated by this kinase, suggesting an alteration of hnRNP K activities. This is the first example of CK2 stimulation by a viral protein in vivo, and we propose that it might facilitate the HSV-1 lytic cycle by, for example, regulating trafficking of ICP27 protein and/or viral RNAs.