Varicella-zoster virus open reading frame 10 protein, the herpes simplex virus VP16 homolog, transactivates herpesvirus immediate-early gene promoters.

The varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein is the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. These are two virion tegument proteins that have extensive amino acid sequence identity in their amino-terminal and middle domains. ORF10, however, lacks the acidic carboxy terminus which is critical for transactivation by VP16. Earlier studies showed that VZV ORF10 does not form a tertiary complex with the TAATGARAT regulatory element (where R is a purine) with which HSV-1 VP16 interacts, suggesting that ORF10 may not have transactivating ability. Using transient-expression assays, we show that VZV ORF10 is able to transactivate VZV immediate-early (IE) gene (ORF62) and HSV-1 IE gene (ICP4 and ICP0) promoters. Furthermore, cell lines stably expressing ORF10 complement the HSV-1 mutant in1814, which lacks the transactivating function of VP16, and enhance the de novo synthesis of infectious virus following transfection of HSV-1 virion DNA. These results indicate that ORF10, like its HSV-1 homolog VP16, is a transactivating protein despite the absence of sequences similar to the VP16 carboxy-terminal domain. The transactivating function of the VZV ORF10 tegument protein may be critical for efficient initiation of viral infection.     

Association of ICP0 but not ICP27 with purified virions of herpes simplex virus type 1.

Recent studies have shown that ICP4, one of the major immediate-early proteins of herpes simplex virus type 1 is present within the tegument region of the virion (F. Yao and R. J. Courtney, J. Virol. 63:3338-3344, 1989). With monoclonal antibodies to two additional immediate-early proteins, ICP0 and ICP27, and Western blot (immunoblot) analysis, ICP0, but not ICP27, was also found to be associated with purified virus particles. In an effort to localize the ICP0 within the virion, purified virions were treated with trypsin in the presence and absence of detergent. The data suggest that ICP0 is located within the tegument region of the virion and is not localized in the envelope or within the nucleocapsid. The number of molecules of ICP0 per virion was estimated to be approximately 150.     

A major transcriptional regulatory protein (ICP4) of herpes simplex virus type 1 is associated with purified virions

Herpes simplex virus type 1 was purified by density gradient centrifugation, and the virion-associated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By Western blot (immunoblot) analysis with an anti-ICP4 monospecific serum, the results indicated that ICP4, one of the five immediate-early proteins of herpes simplex virus type 1, was associated with the purified virions. To define the location of ICP4 within the virion, trypsin digestion experiments were performed. Purified virions were treated with trypsin in the presence or absence of detergent. The virus envelope appeared to protect ICP4 from the trypsin, since virus-associated ICP4 was sensitive to digestion only after detergent treatment. In addition, ICP4 remained associated with the virus particle when the virion-specific glycoproteins were removed after detergent treatment. Finally, ICP4 was not detected in purified preparations of type A and B capsids isolated from the nuclear fraction of virus-infected cells. The above-mentioned data suggest that detectable amounts of ICP4 are present within the tegument region of the virion.     

Evidence that the immediate-early gene product ICP4 is necessary for the genome of the herpes simplex virus type 1 ICP4 deletion mutant strain d120 to circularize in infected cells

Following infection, the physical state of linear herpes simplex virus (HSV) genomes may change into an "endless" or circular form. In this study, using Southern blot analysis of the HSV genome, we provide evidence that immediate-early protein ICP4 is involved in the process of converting the linear HSV-1 ICP4-deleted mutant strain d120 genome into its endless form. Under conditions where de novo viral DNA synthesis was inhibited, the genome of the ICP4 deletion mutant d120 failed to assume an endless conformation following infection of Vero cells (compared with the ability of wild-type strain KOS). This defect was reversed in the Vero-derived cell line E5, which produces the ICP4 protein, suggesting that ICP4 is necessary and sufficient to complement the d120 defect. When ICP4 protein was provided by the replication-defective DNA polymerase mutant HP66, the genomes of mutant d120 could assume an endless conformation in Vero cells. Western blot analysis using antibody specific to the ICP4 protein showed that although the d120 virions contained ICP4 protein, the majority of that ICP4 protein was in a 40-kDa truncated form, with only a small fraction present as a full-length 175-kDa protein. When expression of ICP4 protein from E5 cells was inhibited by cycloheximide, the d120 virion-associated ICP4 protein was unable to mediate endless formation after infection of E5 cells. Collectively, these data suggest that ICP4 protein has an important role in mediating the endless formation of the HSV-1 genome upon infection and that this function can be provided in trans.     

Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis.

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.     

The UL48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions

The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein. UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-DeltaUL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-DeltaUL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.     

Varicella-zoster virus open reading frame 61 protein is functionally homologous to herpes simplex virus type 1 ICP0.

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is thought to be the homolog of herpes simplex virus type 1 (HSV-1) ICP0, based on gene location and limited amino acid homology. However, HSV-1 ICP0 trans activates HSV-1 genes, while VZV ORF61 protein trans represses the function of VZV trans activators on VZV promoters in transient expression assays. To investigate the functional relatedness of HSV-1 ICP0 and VZV ORF61 protein, we established Vero and MeWo cell lines which stably express VZV ORF61 under the control of a metallothionein promoter and performed complementation studies with an HSV-1 ICP0 deletion mutant (7134). Mutant 7134 is impaired for plaque formation and replication at a low multiplicity of infection in cell culture, but these defects were complemented by up to 200-fold in Vero cell lines expressing VZV ORF61. Likewise, the efficiency of plaque formation was improved by up to 100-fold in MeWo cell lines expressing VZV ORF61. A cell line expressing another VZV immediate-early gene product (ORF62) was unable to complement mutant 7134. HSV-1 mutants which are deleted for other HSV-1 immediate-early gene products (ICP4, ICP27) were unable to grow in VZV ORF61-expressing cell lines. These results indicate that, despite marked differences in their sequences and in effects on their cognate promoters in transient expression assays, VZV ORF61 protein is the functional homolog of HSV-1 ICP0.     

A pre-immediate-early role for tegument ICP0 in the proteasome-dependent entry of herpes simplex virus

Herpes simplex virus (HSV) entry requires host cell 26S proteasomal degradation activity at a postpenetration step. When expressed in the infected cell, the HSV immediate-early protein ICP0 has E3 ubiquitin ligase activity and interacts with the proteasome. The cell is first exposed to ICP0 during viral entry, since ICP0 is a component of the inner tegument layer of the virion. The function of tegument ICP0 is unknown. Deletion of ICP0 or mutations in the N-terminal RING finger domain of ICP0 results in the absence of ICP0 from the tegument. We show here that these mutations negatively influenced the targeting of incoming capsids to the nucleus. Inhibitors of the chymotrypsin-like activity of the proteasome the blocked entry of virions containing tegument ICP0, including ICP0 mutants that are defective in USP7 binding. However, ICP0-deficient virions were not blocked by proteasomal inhibitors and entered cells via a proteasome-independent mechanism. ICP0 appeared to play a postpenetration role in cells that supported either endocytosis or nonendosomal entry pathways for HSV. The results suggest that ICP0 mutant virions are defective upstream of viral gene expression at a pre-immediate-early step in infection. We propose that proteasome-mediated degradation of a virion or host protein is regulated by ICP0 to allow efficient delivery of entering HSV capsids to the nuclear periphery.     

Varicella-zoster virus (VZV) open reading frame 10 protein, the homolog of the essential herpes simplex virus protein VP16, is dispensable for VZV replication in vitro.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein in the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. VZV ORF10 transactivates the VZV IE62 gene and is a tegument protein present in the virion. HSV-1 VP16, a potent transactivator of HSV-1 immediate-early genes and tegument protein, is essential for HSV-1 replication in vitro. To determine whether VZV ORF10 is required for viral replication in vitro, we constructed two VZV mutants which were unable to express ORF10. One mutant had a stop codon after the 61st codon of the ORF10 gene, and the other mutant was deleted for all but the last five codons of the gene. Both VZV mutants grew in cell culture to titers similar to that of the parental virus. To determine whether HSV-1 VP16 alters the growth of VZV, we constructed a VZV mutant in which VP16 was inserted in place of ORF10. Using immune electron microscopy, we found that HSV-1 VP16 was present in the tegument of the recombinant VZV virions. The VZV VP16 substitution mutant produced smaller plaques and grew to a lower titer than parental virus. Thus, VZV ORF10 is not required for growth of the virus in vitro, and substitution of HSV-1 VP16 for VZV ORF10 impairs the growth of VZV.     

The varicella-zoster virus immediate-early protein IE62 is a major component of virus particles.

Varicella-zoster virus (VZV) open reading frame (ORF) 62 potentially encodes a protein with considerable amino acid homology to the herpes simplex virus (HSV) immediate-early regulatory polypeptide ICP4 (or IE3). To identify and characterize its protein product(s) (IE62), we used a rabbit antiserum prepared against a synthetic peptide corresponding to the C-terminal 13 amino acids of the predicted protein. This antiserum reacted with phosphorylated polypeptides of 175 to 180 kDa that were made in VZV-infected cells and in cells infected with a vaccinia virus recombinant expressing IE62, but not in control-infected cells, confirming its specificity and reactivity to the IE62 protein. The antiserum recognized a 175-kDa polypeptide in purified virions that comigrated with a major structural protein. Comparison of this reactivity with that of an antipeptide antiserum directed against the VZV ORF 10 product (homologous to the HSV major structural protein VP16) indicates similar levels of ORF 62 and ORF 10 polypeptides in VZV virions. In contrast, antipeptide antiserum directed against the VZV ORF 29 product, the homolog of the HSV major DNA-binding protein, failed to recognize any protein in our virion preparations. Treatment of virions with detergents that disrupt the virion envelope did not dissociate IE62 from the nucleocapsid-tegument structure of the virion. Differential sensitivity of VZV virion IE62 to trypsin digestion in the presence or absence of Triton X-100 indicates that IE62 is protected from trypsin degradation by the virus envelope; since it is not a nucleocapsid protein, we conclude that it is part of the tegument. Finally, we show that the virion 175-kDa protein either can autophosphorylate or is a major substrate in vitro for virion-associated protein kinase activity.     

The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.     

Varicella-zoster virus open reading frame 4 protein is functionally distinct from and does not complement its herpes simplex virus type 1 homolog, ICP27.

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a putative immediate-early protein which is homologous to herpes simplex virus type 1 (HSV-1) ICP27 on the basis of gene location and similarity in amino acid sequence. In transient expression assays, however, ORF4 and ICP27 exhibit different properties. ICP27 alone has little activity on target plasmids, but it acts as a transactivator or a transrepressor in the presence of other HSV-1 transactivators. In contrast, ORF4 directly transactivates plasmids containing homologous or heterologous promoters and has no apparent transrepressing activity. To further illuminate the functional similarities and differences between ORF4 and ICP27, Vero cell lines which express ORF4 under the inducible metallothionein promoter were constructed. Cell lines expressing functionally active ORF4 protein upregulated the expression of transfected VZV target plasmids but were unable to efficiently complement HSV-1 ICP27 mutants. These results indicate that, despite structural similarities, VZV ORF4 and HSV-1 ICP27 behave differently in transient expression assays and may play different roles in virus replication.