Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant.

Herpes simplex virus (HSV) encodes a ribonucleotide reductase consisting of two subunits (140 and 38 kilodaltons) whose genes map to coordinates 0.56 to 0.60 on the viral genome. Host cell lines containing the HpaI F fragment which includes the reductase subunit genes of HSV type 1 strain KOS (coordinates 0.535 to 0.620) were generated. Transfection of these cells with a plasmid containing the immediate-early ICP0 gene resulted in the expression of ICP6; interestingly, ICP4 plasmids failed to induce expression, indicating an unusual pattern of ICP6 regulation. One such cell line (D14) was used to isolate a mutant with the structural gene of lacZ inserted into the ICP6 gene such that the lacZ gene is read in frame with the N-terminal region of ICP6. This mutant generated a protein containing 434 amino acids (38%) of the N terminus of ICP6 fused to beta-galactosidase under control of the endogenous ICP6 promoter. Screening for virus recombinants was greatly facilitated by staining virus plaques with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside (X-gal). Enzyme assays of infected BHK cells indicated that the mutant is incapable of inducing viral ribonucleotide reductase activity. Surprisingly, although plaque size was greatly reduced, mutant virus yield was reduced only four- to fivefold compared with that of the wild type grown in exponentially growing Vero cells. Mutant virus plaque size, yields, and ability to synthesize viral DNA were more severely compromised in serum-starved cells as compared with the wild type grown under the same condition. Although our evidence suggests that the HSV type 1 ribonucleotide reductase is not required for virus growth and DNA replication in dividing cells, it may be required for growth in nondividing cells.     

HSV‐1 ICP27 targets the TBK1‐activated STING signalsome to inhibit virus‐induced type I IFN expression

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS–STING–TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV‐1 inhibits IFN expression in this cell type. Here, we show that HSV‐1 inhibits type I IFN induction through the cGAS–STING–TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV‐1 inhibits expression of type I IFN in human macrophages through ICP27‐dependent targeting of the TBK1‐activated STING signalsome.     

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.     

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 cloned DNA fragments induce coumermycin A1 resistance in Escherichia coli.

We have taken a new approach to identify and fine map previously undescribed herpes simplex virus (HSV) functions. In experiments described in this report the antibiotic coumermycin A1 was used to select two HSV type 1 BamHI fragments cloned in pBR322 that confer partial resistance to drug-susceptible Escherichia coli. The genes encoding these HSV functions have been designated cour-1 and cour-2 and have been fine mapped to the HSV sequences. HSV-cour1 is located at the left end of BamHI-F near HSV type 1 genomic map coordinate 0.645. cour-2 maps to BamHI-M', which is a 159-base-pair internal component of the alpha ICP4-coding sequence located in the reiterated sequences of the S component. In pBR322 both inserts apparently rely on the tet promoter for expression. Additionally, cour-2 functions when present as a BamHI insert in pUC7. The analysis of cour-2 "maxi" cell proteins reveals the presence of proteins produced by the fusion of HSV-1 BamHI-M' sequences and the sequences of the vector genes, i.e., the major tet product for pBR322 and the modified beta-galactosidase for pUC7. These data suggest that the development of bacterial assays for fusion products of eucaryotic DNA open reading frames in plasmid vectors may be a useful technique for initiating gene function studies.     

Efficient activation of viral genomes by levels of herpes simplex virus ICP0 insufficient to affect cellular gene expression or cell survival

Herpes simplex virus (HSV) ICP0 can effectively activate gene expression from otherwise silent promoters contained on persisting viral genomes. However, the expression of high levels of ICP0, as from ICP4(-) HSV type 1 (HSV-1) vectors, results in marked toxicity. We have analyzed the results of ICP0 expressed from an E1(-) E4(-) adenovirus vector (AdS.11E4ICP0) in which ICP0 expression is controlled from the endogenous adenoviral E4 promoter. In this system, the expression level of ICP0 was reduced more than 1,000-fold relative to the level of expression from HSV-1 vectors. This low level of ICP0 did not affect cellular division or greatly perturb cellular metabolism as assessed by gene expression array analysis comparing the effects of HSV and adenovirus vector strains. However, this amount of ICP0 was sufficient to quantitatively destroy ND10 structures as measured by promyelocytic leukemia immunofluorescence. The levels of adenovirus-expressed ICP0 were sufficient to activate quiescent viral genomes in trans and promote persistent transgene expression in cis. Moreover, infection of complementing cells with AdS.11E4ICP0 promoted viral growth and resulted in a 20-fold increase in the plaquing efficiency of d109, a virus defective for all five immediate-early genes. Thus, the low level expression of ICP0 from the E1(-) E4(-) adenovirus vector may increase the utility of adenovirus vectors and also provides a means to efficiently quantify and possibly propagate HSV vectors defective in ICP0. Importantly, the results demonstrate that the activation function of ICP0 may not result from changes in cellular gene expression, but possibly as a direct consequence of an enzymatic function inherent to the protein that may involve its action at ND10 resulting in the preferential activation of viral genomes.     

Requirements for the induction of interleukin-6 by herpes simplex virus-infected leukocytes

Cytokines play important roles in the clearance of herpes simplex virus (HSV) infections and in virus-induced immunopathology. One cytokine known to contribute to resistance against HSV is interleukin-6 (IL-6). Here we have investigated virus-cell interactions responsible for IL-6 induction by HSV in leukocytes. Both HSV type 1 and type 2 are potent inducers of IL-6, and this phenomenon is augmented in the presence of gamma interferon. The ability to induce IL-6 is dependent on de novo protein synthesis and is sensitive to UV irradiation of the virus. Virus mutants lacking the virion-transactivating protein VP16 or any of the immediate-early proteins ICP0, ICP4, or ICP27 displayed unaltered capacities to induce IL-6. However, wild-type virus was unable to induce IL-6 in a macrophage cell line overexpressing a mutant of double-stranded RNA-activated protein kinase (PKR). This suggests a role for PKR in HSV-induced IL-6 expression. HSV infection led to enhanced binding to the kappaB, CRE, and AP-1 sites of the IL-6 promoter, and inhibitors against NF-kappaB and the p38 kinase strongly reduced accumulation of IL-6 mRNA in infected cells. Moreover, macrophage cell lines expressing dominant negative mutants of IkappaBalpha and p38 responded to HSV-1 infection with reduced IL-6 expression compared to the control-vector-transfected cell line. The results show that induction of IL-6 by HSV in leukocytes is dependent on PKR and cellular signaling through NF-kappaB and a p38-dependent pathway.     

Properties of herpes simplex virus DNA polymerase and characterization of its associated exonuclease activity.

Herpes simplex virus (HSV) DNA polymerase was isolated on a large-scale from African green monkey kidney cells infected with HSV type 1 (HSV-1) strain Angelotti. After DNA-cellulose chromatography the enzyme showed a specific activity of 48,000 units/mg protein. Three major single polypeptides with molecular weights of 144,000, 74,000 and 29,000 were copurified with the enzyme activity at the DNA-cellulose ste. By its chromatographic behavior and by template studies, the HSV DNA polymerase activity was clearly distinguishable from cellular alpha, beta and gamma DNA polymerase activities. Two exonucleolytic activities were found in the DNA-cellulose enzyme preparation. The main exonucleolytic activity, which degraded both single-stranded and double-stranded DNA to deoxynucleoside 5'-monophosphates, was separated by subsequent velocity sedimentation. The remaining exonucleolytic activity was not separable from the HSV DNA polymerase by several chromatographic steps and by velocity sedimentation at high ionic strength. This novel exonuclease and HSV DNA polymerase were equally sensitive both to phosphonoacetic acid and Zn2+ ions, inhibitors of the viral polymerase. Similar to the 3'-to-5'-exonuclease of procaryotic DNA polymerases and mammalian DNA polymerase delta, the HSV-polymerase-associated exonuclease catalyzed the removal of 3'-terminal nucleotides from the primer/template as well as the template-dependent conversion of deoxynucleoside triphosphates to monophosphates.     

Aanlysis of dCMP deaminase and CDP reductase levels in hamster cells infected by herpes simplex virus.

Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity.     

Herpes simplex virus ICP27 is required for virus-induced stabilization of the ARE-containing IEX-1 mRNA encoded by the human IER3 gene

Herpes simplex virus (HSV) stifles cellular gene expression during productive infection of permissive cells, thereby diminishing host responses to infection. Host shutoff is achieved largely through the complementary actions of two viral proteins, ICP27 and virion host shutoff (vhs), that inhibit cellular mRNA biogenesis and trigger global mRNA decay, respectively. Although most cellular mRNAs are thus depleted, some instead increase in abundance after infection; perhaps surprisingly, some of these contain AU-rich instability elements (AREs) in their 3'-untranslated regions. ARE-containing mRNAs normally undergo rapid decay; however, their stability can increase in response to signals such as cytokines and virus infection that activate the p38/MK2 mitogen-activated protein kinase (MAPK) pathway. We and others have shown that HSV infection stabilizes the ARE mRNA encoding the stress-inducible IEX-1 mRNA, and a previous report from another laboratory has suggested vhs is responsible for this effect. However, we now report that ICP27 is essential for IEX-1 mRNA stabilization whereas vhs plays little if any role. A recent report has documented that ICP27 activates the p38 MAPK pathway, and we detected a strong correlation between this activity and stabilization of IEX-1 mRNA by using a panel of HSV type 1 (HSV-1) isolates bearing an array of previously characterized ICP27 mutations. Furthermore, IEX-1 mRNA stabilization was abrogated by the p38 inhibitor SB203580. Taken together, these data indicate that the HSV-1 immediate-early protein ICP27 alters turnover of the ARE-containing message IEX-1 by activating p38. As many ARE mRNAs encode proinflammatory cytokines or other immediate-early response proteins, some of which may limit viral replication, it will be of great interest to determine if ICP27 mediates stabilization of many or all ARE-containing mRNAs.     

Equine Herpesvirus 1 Gene 12 Can Substitute for vmw65 in the Growth of Herpes Simplex Virus (HSV) Type 1, Allowing the Generation of Optimized Cell Lines for the Propagation of HSV Vectors with Multiple Immediate-Early Gene Defects

Herpes simplex virus (HSV) has often been suggested for development as a vector, particularly for the nervous system. Considerable evidence has shown that for use of HSV as a vector, immediate-early (IE) gene expression must be minimized or abolished, otherwise such vectors are likely to be highly cytotoxic. Mutations of vmw65 which abolish IE promoter transactivating activity may also be included to reduce IE gene expression generally. However, when vmw65 mutations are combined with an IE gene deletion, such viruses are hard to propagate, even on cells which otherwise complement the IE gene deletion effectively. We have found that vmw65 mutants can be effectively grown on cell lines expressing equine herpesvirus 1 gene 12, a non-HSV homologue of vmw65 with little sequence similarity to its HSV counterpart. This prevents repair by homologous recombination of vmw65 mutations in the virus, which would occur if mutations were complemented by vmw65 itself. The gene 12 protein is not packaged into HSV virions, which is important if viruses grown on such cells are to be used as vectors. These results not only further strengthen the evidence for direct functional homology between and similar modes of action of the two proteins but have allowed the generation of gene 12-containing cell lines in which ICP4 and ICP27 expression is induced by virus infection (probably by ICP0) and which give efficient growth of viruses deficient in ICP27, ICP4, and vmw65 (the viruses also have ICP34.5/ORFP deleted). Efficient growth of such viruses has not previously been possible. As these viruses are highly deficient in IE gene expression generally, such virus-cell line combinations may provide an alternative to HSV vectors with deletions of all four of the regulatory IE genes which, for optimal growth, require cell lines containing all four IE genes but which are hard to generate due to the intrinsic cytotoxicity of each of the proteins.     

Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.     

Vaccination with recombinant vaccinia viruses expressing ICP27 induces protective immunity against herpes simplex virus through CD4+ Th1+ T cells.

This study was designed to evaluate the efficacy and mechanisms of protection mediated by recombinant vaccinia viruses encoding immediate-early (IE) proteins of herpes simplex virus type 2 (HSV-2). Three mouse strains were immunized against the IE proteins ICP27, ICP0, and ICP4, and mice were challenged intracutaneously in the zosteriform model with HSV-2 strain MS. Protection was observed only following immunization with the ICP27 construct and then only in the BALB/c mouse strain. Protection in BALB/c mice was ablated by CD4+ T-cell suppression but remained intact in animals depleted of CD8+ T cells. Moreover, protection could be afforded to SCID nude recipients with CD4+ but not CD8+ T cells from ICP27-immunized mice. Only BALB/c mice developed a delayed-type hypersensitivity reaction to HSV-2, and in vitro measurements of humoral and cell-mediated immunity revealed response patterns to ICP27 and HSV that differed between protected BALB/c and unprotected mouse strains. Accordingly, BALB/c responses showed antigen-induced cytokine profiles dominated by type 1 cytokines, whereas C57BL/6 and C3H/HeN mice generated cytokine responses mainly of the type 2 variety. Our results may indicate that protection against zosterification is mainly mediated by CD4+ T cells that express a type 1 cytokine profile and that protective vaccines against HSV which effectively induce such T-cell responses should be chosen.     

Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 X HSV-2 recombinants.

In an earlier paper (Morse et al., J. Virol 24:231--248, 1977) we reported on the provenance of the DNA sequences in 26 herpes simplex virus type 1 (HSV-1) X HSV-2 recombinants as determined from analyses of their DNAs with at least five restriction endonucleases. This report deals with the polypeptides specified by the recombinants and by their HSV-1 and HSV-2 parents. We have identified (i) the corresponding HSV-1 and HSV-2 polypeptides with molecular weights ranging from 20,000 to more than 200,000, (ii) the polypeptides that undergo rapid post-translational processing, and (iii) polypeptides that vary intratypically in apparent molecular weight. By comparing the segregation patterns of the polypeptides with those of the DNA sequence of the recombinants, we have mapped the templates specifying 26 polypeptides and several viral functions on the physical map of HSV DNA. The data show the following: (i) alpha polypeptides map at the termini of the L and S components of the HSV DNA. Although alpha ICP 27 maps entirely within the reiterated region of the L component, the template for alpha ICP 4 may lie only in part within the reiterated sequences of the S component. Of note is the finding that cells infected with a recombinant that contains both HSV-1 and HSV-2 DNA sequences in the S component produced alpha ICP 4 of both HSV-1 and HSV-2. (ii) Templates specifying beta and gamma polypeptides map in the L component and appear to be randomly distributed. (iii) Thymidine kinase and resistance to phosphonoacetic acid mapped in the L component. In addition, we have taken advantage of the rapid inhibition of host protein synthesis characteristic of HSV-2 infections and syncytial plaque morphology to also map the template(s) responsible for these functions in the L component. The implications of the template arrangement in HSV DNA are discussed.     

Herpes simplex virus requires poly(ADP-ribose) polymerase activity for efficient replication and induces extracellular signal-related kinase-dependent phosphorylation and ICP0-dependent nuclear localization of tankyrase 1

Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) which localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Poly(ADP-ribosyl)ation of the nuclear mitotic apparatus (NuMA) protein by tankyrase 1 during mitosis is essential for sister telomere resolution and mitotic spindle pole formation. In interphase cells, tankyrase 1 resides in the cytoplasm, and its role therein is not well understood. In this study, we found that herpes simplex virus (HSV) infection induced extensive modification of tankyrase 1 but not tankyrase 2. This modification was dependent on extracellular signal-regulated kinase (ERK) activity triggered by HSV infection. Following HSV-1 infection, tankyrase 1 was recruited to the nucleus. In the early phase of infection, tankyrase 1 colocalized with ICP0 and thereafter localized within the HSV replication compartment, which was blocked in cells infected with the HSV-1 ICP0-null mutant R7910. In the absence of infection, ICP0 interacted with tankyrase 1 and efficiently promoted its nuclear localization. HSV did not replicate efficiently in cells depleted of both tankyrases 1 and 2. Moreover, XAV939, an inhibitor of tankyrase PARP activity, decreased viral titers to 2 to 5% of control values. We concluded that HSV targets tankyrase 1 in an ICP0- and ERK-dependent manner to facilitate its replication.