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.     

Identification and characterization of the herpes simplex virus type 1 protein encoded by the UL37 open reading frame

The UL37 open reading frame of the herpes simplex virus type 1 (HSV-1) DNA genome is located between map units 0.527 and 0.552. We have identified and characterized the UL37 protein product in HSV-1-infected cells. The presence of the UL37 protein was detected by using a polyclonal rabbit antiserum directed against an in vitro-translated product derived from an in vitro-transcribed UL37 mRNA. The UL37 open reading frame encodes for a protein with an apparent molecular mass of 120 kDa in HSV-1-infected cells; the protein's mass was assigned on the basis of its migration in sodium dodecyl sulfate-polyacrylamide gels. The UL37 protein is not present at detectable levels in purified HSV-1 virions, suggesting that it is not a structural protein. Analysis of time course experiments and experiments using DNA synthesis inhibitors demonstrated that the UL37 protein is expressed prior to the onset of viral DNA synthesis, reaching maximum levels late in infection, classifying it as a gamma 1 gene. Elution of HSV-1-infected cell proteins from single-stranded DNA agarose columns by using a linear KCl gradient demonstrated that the UL37 protein elutes from this matrix at a salt concentration similar to that observed for ICP8, the major HSV-1 DNA-binding protein. In addition, computer-assisted analysis revealed a potential ATP-binding domain in the predicted UL37 amino acid sequence. On the basis of the kinetics of appearance and DNA-binding properties, we hypothesize that UL37 represents a newly recognized HSV-1 DNA-binding protein that may be involved in late events in viral replication.     

Properties of the protein encoded by the UL32 open reading frame of herpes simplex virus 1.

The functions previously assigned to the essential herpes simplex virus 1 UL32 protein were in cleavage and/or packaging of viral DNA and in maturation and/or translocation of viral glycoproteins to the plasma membrane. The amino acid sequence predicts N-linked glycosylation sites and sequences conserved in aspartyl proteases and in zinc-binding proteins. We report the following. (i) The 596-amino-acid UL32 protein accumulated predominantly in the cytoplasm of infected cells but was not metabolically labeled with glucosamine and did not band with membranes containing a known glycoprotein in flotation sucrose density gradients. The UL32 protein does not, therefore, have the properties of an intrinsic membrane protein. (ii) Experiments designed to demonstrate aspartyl protease activity in a phage display system failed to reveal proteolytic activity. Moreover, substitution of Asp-110 with Gly in the sequence Asp-Thr-Gly, the hallmark of aspartyl proteases, had no effect on viral replication in Vero and SK-N-SH cell lines or in human foreskin fibroblasts. Therefore, if the UL32 protein functions as a protease, this function is not required in cells in culture. (iii) Both the native UL32 protein and a histidine-tagged UL32 protein made in recombinant baculovirus-infected insect cells bound zinc. The consensus sequence is conserved in the UL32 homologs from varicella-zoster virus and equine herpesvirus 1. UL32 protein is therefore a cysteine-rich, zinc-binding essential cytoplasmic protein whose function is not yet clear.     

Identification and characterization of the herpes simplex virus type 1 virion protein encoded by the UL35 open reading frame.

The UL35 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) has been predicted from DNA sequence analysis to encode a small polypeptide with a molecular weight of 12,095. We have investigated the protein product of the UL35 ORF by using a trpE-UL35 gene fusion to produce a corresponding fusion protein in Escherichia coli. The TrpE-UL35 chimeric protein was subsequently isolated and used as a source of immunogen for the production of rabbit polyclonal antiserum directed against the UL35 gene product. The TrpE-UL35 antiserum was found to recognize a 12-kDa protein which was specifically present in HSV-1-infected cells. By utilizing the TrpE-UL35 antiserum, the kinetics of synthesis of the UL35 gene product was examined, and these studies indicate that UL35 is expressed as a gamma 2 (true late) gene. The 12-kDa protein recognized by the TrpE-UL35 antiserum was associated with purified HSV-1 virions and type A and B capsids, suggesting that the UL35 ORF may encode the 12-kDa capsid protein variably designated p12, NC7, or VP26. To confirm this assignment, immunoprecipitation and immunoblotting studies were performed to demonstrate that the TrpE-UL35 antiserum reacts with the same polypeptide as an antiserum directed against the purified p12 capsid protein (anti-NC7) (G.H. Cohen, M. Ponce de Leon, H. Diggelmann, W.C. Lawrence, S.K. Vernon, and R.J. Eisenberg, J. Virol. 34:521-531, 1980). Furthermore, the anti-NC7 serum was also found to react with the TrpE-UL35 chimeric protein isolated from E. coli, providing additional evidence that the UL35 gene encodes p12. On the basis of these studies, we conclude that UL35 represents a true late gene which encodes the 12-kDa capsid protein of HSV-1.     

Binding partners for the UL11 tegument protein of herpes simplex virus type 1

The product of the U(L)11 gene of herpes simplex virus type 1 (HSV-1) is a 96-amino-acid tegument protein that accumulates on the cytoplasmic face of internal membranes. Although it is thought to be important for nucleocapsid envelopment and egress, the actual function of this protein is unknown. Previous studies focused on the characterization of sequence elements within the UL11 protein that function in membrane binding and trafficking to the Golgi apparatus. Binding was found to be mediated by two fatty acyl groups (myristate and palmitate), while an acidic cluster and a dileucine motif were identified as being important for the recycling of UL11 from the plasma membrane to the Golgi apparatus. The goal of the experiments described here was to identify and characterize binding partners (viral or cellular) of UL11. Using both immunoprecipitation and glutathione S-transferase (GST) pull-down assays, we identified a 40-kDa protein that specifically associates with UL11 from infected Vero cells. Mutational analyses revealed that the acidic cluster and the dileucine motif are required for this association, whereas the entire second half of UL11 is not. In addition, UL11 homologs from pseudorabies and Marek's disease herpesviruses were also found to be capable of binding to the 40-kDa protein from HSV-1-infected cells, suggesting that the interaction is conserved among alphaherpesviruses. Purification and analysis of the 40-kDa protein by mass spectrometry revealed that it is the product of the U(L)16 gene, a virion protein reported to be involved in nucleocapsid assembly. Cells transfected with a UL16-green fluorescent protein expression vector produced a protein that was of the expected size, could be pulled down with GST-UL11, and accumulated in a Golgi-like compartment only when coexpressed with UL11, indicating that the interaction does not require any other viral products. These data represent the first steps toward elucidating the network of tegument proteins that UL11 links to membranes.     

Packaging determinants in the UL11 tegument protein of herpes simplex virus type 1

The UL11 gene of herpes simplex virus type 1 encodes a 96-amino-acid tegument protein that is myristylated, palmitylated, and phosphorylated and is found on the cytoplasmic faces of nuclear, Golgi apparatus-derived, and plasma membranes of infected cells. Although this protein is thought to play a role in virus budding, its specific function is unknown. Purified virions were found to contain approximately 700 copies of the UL11 protein per particle, making it an abundant component of the tegument. Moreover, comparisons of cell-associated and virion-associated UL11 showed that packaging is selective for underphosphorylated forms, as has been reported for several other tegument proteins. Although the mechanism by which UL11 is packaged is unknown, previous studies have identified several sequence motifs in the protein that are important for membrane binding, intracellular trafficking, and interaction with UL16, another tegument protein. To ascertain whether any of these motifs are needed for packaging, a transfection/infection-based assay was used in which mutant forms of the protein must compete with the wild type. In this assay, the entire C-terminal half of UL11 was found to be dispensable. In the N-terminal half, the sites of myristylation and palmitylation, which enable membrane-binding and Golgi apparatus-specific targeting, were found to be essential for efficient packaging. The acidic cluster motif, which is not needed for Golgi apparatus-specific targeting but is involved in recycling the protein from the plasma membrane and for the interaction with UL16, was found to be essential, too. Thus, something other than mere localization of UL11 to Golgi apparatus-derived membranes is needed for packaging. The critical factor is unlikely to be the interaction with UL16 because other mutants that fail to bind this protein (due to removal of the dileucine-like motif or substitutions with foreign acidic clusters) were efficiently packaged. Collectively, these results suggest that UL11 packaging is not driven by a passive mechanism but instead requires trafficking through a specific pathway.     

Characterization of a temperature-sensitive mutant of the UL15 open reading frame of herpes simplex virus 1.

The UL15 gene of herpes simplex virus 1 consists of two exons and is highly conserved among the herpesviruses sequenced to date. Other than its homology to a phage protein involved in the packaging of DNA, nothing is known of its function. This report concerns the isolation of a temperature-sensitive mutant with a mutation mapping in the UL15 open reading frame. Cells infected with the parent, mutant, and rescued viruses all make DNA at the nonpermissive temperature. Direct analyses of the DNA and electron microscopic studies indicate that although viral DNA is made, it is not packaged into capsids present in nuclei. These studies suggest that UL15 may be involved in the packaging of viral DNA.     

The UL36 tegument protein of herpes simplex virus 1 has a composite binding site at the capsid vertices

Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope. The tegument comprises at least 20 proteins destined for delivery into the host cell. As the tegument does not have a regular structure, the question arises of how its proteins are recruited. The herpes simplex virus 1 (HSV-1) tegument is known to contact the capsid at its vertices, and two proteins, UL36 and UL37, have been identified as candidates for this interaction. We show that the interaction is mediated exclusively by UL36. HSV-1 nucleocapsids extracted from virions shed their UL37 upon incubation at 37°C. Cryo-electron microscopy (cryo-EM) analysis of capsids with and without UL37 reveals the same penton-capping density in both cases. As no other tegument proteins are retained in significant amounts, it follows that this density feature (～100 kDa) represents the ordered portion of UL36 (336 kDa). It binds between neighboring UL19 protrusions and to an adjacent UL17 molecule. These observations support the hypothesis that UL36 plays a major role in the tegumentation of the virion, providing a flexible scaffold to which other tegument proteins, including UL37, bind. They also indicate how sequential conformational changes in the maturing nucleocapsid control the ordered binding, first of UL25/UL17 and then of UL36.     

The size and symmetry of B capsids of herpes simplex virus type 1 are determined by the gene products of the UL26 open reading frame.

Herpes simplex virus type 1 (HSV-1) B capsids are composed of seven proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 in order of decreasing molecular weight. Three proteins (21, 22a, and VP24) are encoded by a single open reading frame (ORF), UL26, and include a protease whose structure and function have been studied extensively by other investigators. The protease encoded by this ORF generates VP24 (amino acids 1 to 247), a structural component of the capsid and mature virions, and 21 (residues 248 to 635). The protease also cleaves C-terminal residues 611 to 635 of 21 and 22a, during capsid maturation. Protease activity has been localized to the N-terminal 247 residues. Protein 22a and probably the less abundant protein 21 occupy the internal volume of capsids but are not present in virions; therefore, they may form a scaffold that is used for B capsid assembly. The objective of the present study was to isolate and characterize a mutant virus with a null mutation in UL26. Vero cells were transformed with plasmid DNA that encoded ORF UL25 through UL28 and screened for their ability to support the growth of a mutant virus with a null mutation in UL27 (K082). Four of five transformants that supported the growth of the UL27 mutant also supported the growth of a UL27-UL28 double mutant. One of these transformants (F3) was used to isolate a mutant with a null mutation in UL26. The UL26 null mutation was constructed by replacement of DNA sequences specifying codons 41 through 593 with a lacZ reporter cassette. Permissive cells were cotransfected with plasmid and wild-type virus DNA, and progeny viruses were screened for their ability to grow on F3 but not Vero cells. A virus with these growth characteristics, designated KUL26 delta Z, that did not express 21, 22a, or VP24 during infection of Vero cells was isolated. Radiolabeled nuclear lysates from infected nonpermissive cells were layered onto sucrose gradients and subjected to velocity sedimentation. A peak of radioactivity for KUL26 delta Z that sedimented more rapidly than B capsids from wild-type-infected cells was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gradient fractions showed that the peak fractions contained VP5, VP19C, VP23, and VP26. Analysis of sectioned cells and of the peak fractions of the gradients by electron microscopy revealed sheet and spiral structures that appear to be capsid shells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of the phosphorylation sites of herpes simplex virus type 1 regulatory protein ICP27

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a 63-kDa phosphoprotein required for viral replication. ICP27 has been shown to contain both stable phosphate groups and phosphate groups that cycle on and off during infection (K. W. Wilcox, A. Kohn, E. Sklyanskaya, and B. Roizman, J. Virol. 33:167-182, 1980). Despite extensive genetic analysis of the ICP27 gene, there is no information available about the sites of the ICP27 molecule that are phosphorylated during viral infection. In this study, we mapped several of the phosphorylation sites of ICP27 following in vivo radiolabeling. Phosphoamino acid analysis showed that serine is the only amino acid that is phosphorylated during infection. Two-dimensional phosphopeptide mapping showed a complex tryptic phosphopeptide pattern with at least four major peptides and several minor peptides. In addition, ICP27 purified from transfected cells yielded a similar phosphopeptide pattern, suggesting that cellular kinases phosphorylate ICP27 during viral infection. In vitro labeling showed that protein kinase A (PKA), PKC, and casein kinase II (CKII) were able to differentially phosphorylate ICP27, resulting in distinct phosphopeptide patterns. The major phosphorylation sites of ICP27 appeared to cluster in the N-terminal portion of the protein, such that a frameshift mutant that encodes amino acids 1 to 163 yielded a phosphopeptide pattern very similar to that seen with the wild-type protein. Further, using small deletion and point mutations in kinase consensus sites, we have elucidated individual serine residues that are phosphorylated in vivo. Specifically, the serine at residue 114 was highly phosphorylated by PKA and the serine residues at positions 16 and 18 serve as targets for CKII phosphorylation in vivo. These kinase consensus site mutants were still capable of complementing the growth of an ICP27-null mutant virus. Interestingly, phosphorylation of the serine at residue 114, which lies within the major nuclear localization signal, appeared to modulate the efficiency of nuclear import of ICP27.     

Analysis of the interaction between the UL11 and UL16 tegument proteins of herpes simplex virus

The UL11 and UL16 tegument proteins of herpes simplex virus are conserved throughout the herpesvirus family. Previous studies have shown that these proteins interact, perhaps to link UL16-bound nucleocapsids to UL11, which resides on the cytoplasmic face of the trans-Golgi network, where maturation budding occurs. Little is known about the interaction except that it requires the leucine-isoleucine (LI) and acidic cluster motifs in UL11 and that no other viral proteins are involved. In particular, the important question of whether these two proteins bind to each other directly has not been addressed. Accordingly, UL11 and UL16 were expressed in bacteria, and the purified proteins were found to retain the ability to interact in a manner that was dependent upon the LI and acidic cluster. In an attempt to map the UL11-binding site contained in UL16, a large number of deletion mutants were constructed. The first 40 (nonconserved) amino acids were found to be dispensable, but all the other constructs failed to bind UL11 or had poor expression in transfected cells, suggesting that UL16 is very sensitive to alterations and probably lacks a multidomain structure. As an alternative strategy for identifying residues that are important for the interaction, the cysteines of UL16 were investigated, because many of these are highly conserved. Approximately half of the 20 cysteines in UL16 have been shown to be covalently modified by N-ethylmaleimide, and this treatment was found to block the interaction with UL11. Moreover, individual serine replacements of six of the most conserved cysteine residues were made, and four of these disrupted the interaction with UL11 without affecting protein stability. However, the UL11-UL16 interaction does not involve the formation of interspecies disulfide bonds, because binding occurred even when all the cysteines in UL11 were eliminated. Thus, UL16 directly interacts with UL11 and does so in a manner that requires free cysteines.     

Pseudorabies virus UL36 tegument protein physically interacts with the UL37 protein

The UL36 open reading frame encoding the tegument protein ICP1/2 represents the largest open reading frame in the genome of herpes simplex virus type 1 (HSV-1). Polypeptides homologous to the HSV-1 UL36 protein are present in all subfamilies of HERPESVIRIDAE: We sequenced the UL36 gene of the alphaherpesvirus pseudorabies virus (PrV) and prepared a monospecific polyclonal rabbit antiserum against a bacterial glutathione S-transferase (GST)-UL36 fusion protein for identification of the protein. The antiserum detected a >300-kDa protein in PrV-infected cells and in purified virions. Interestingly, in coprecipitation analyses using radiolabeled infected-cell extracts, the anti-UL36 serum reproducibly coprecipitated the UL37 tegument protein, and antiserum directed against the UL37 protein coprecipitated the UL36 protein. This physical interaction could be verified using yeast two-hybrid analysis which demonstrated that the UL37 protein interacts with a defined region within the amino-terminal part of the UL36 protein. By use of immunogold labeling, capsids which accumulate in the cytoplasm in the absence of the UL37 protein (B. G. Klupp, H. Granzow, E. Mundt, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) as well as wild-type intracytoplasmic and extracellular virions were decorated by the anti-UL36 antiserum, whereas perinuclear primary enveloped virions were not. We postulate that the physical interaction of the UL36 protein, which presumably constitutes the innermost layer of the tegument (Z. Zhou, D. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210-3218, 1999), with the UL37 protein is an important early step in tegumentation during virion morphogenesis in the cytoplasm.     

Dynamic interactions of the UL16 tegument protein with the capsid of herpes simplex virus

The UL16 tegument protein of herpes simplex virus is conserved throughout the herpesvirus family. It has been reported to be capsid associated and may be involved in budding by providing an interaction with the membrane-bound UL11 protein. UL16 has been shown to be present in all the major locations that capsids are found (i.e., the nucleus, cytoplasm, and virions), but whether it is actually capsid associated in each of these has not been reported. Therefore, capsids were purified from each compartment, and it was found that UL16 was present on cytoplasmic but not nuclear capsids. In extracellular virions, the majority of UL16 (87%) was once again not capsid associated, which suggests that the interaction is transient during egress. Because herpes simplex virus (HSV) buds into the acidic compartment of the trans-Golgi network (TGN), the effect of pH on the interaction was examined. The amount of capsid-associated UL16 dramatically increased when extracellular virions were exposed to mildly acidic medium (pH 5.0 to 5.5), and this association was fully reversible. After budding into the TGN, capsid and tegument proteins also encounter an oxidizing environment, which is conducive to disulfide bond formation. UL16 contains 20 cysteines, including five that are conserved within a putative zinc finger. Any free cysteines that are involved in the capsid interaction or release mechanism of UL16 would be expected to be modified by N-ethylmaleimide, and, consistent with this, the amount of capsid-associated UL16 dramatically increased when virions were incubated with this compound. Taken together, these data suggest a transient interaction between UL16 and capsids, possibly modified in the acidic compartment of secretory vesicles and requiring a release mechanism that involves cysteines.