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.     

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.     

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.     

A specific subform of the human cytomegalovirus transactivator protein pUL69 is contained within the tegument of virus particles.

The polypeptide encoded by the open reading frame UL69 of human cytomegalovirus (HCMV), which is homologous to the immediate-early regulator ICP27 of herpes simplex virus, has recently been identified as a transactivator protein that exerts a broad stimulatory effect on gene expression (M. Winkler, S. A. Rice, and T. Stamminger, J. Virol. 68:3943-3954, 1994). Here, we provide evidence that pUL69 is a phosphorylated tegument protein of HCMV. This finding could be demonstrated by Western blot (immunoblot) analyses with purified virions and a specific antiserum against pUL69. These experiments revealed that one phosphorylated subform of the three pUL69 polypeptides that are synthesized in infected fibroblast cells is contained within the HCMV virion. After the treatment of purified virions with detergents, pUL69 could not be detected within the membrane fraction, suggesting that it is either a capsid or a tegument protein. Its presence within dense bodies, however, shows that pUL69 is a constituent of the viral tegument.     

Cooperativity among herpes simplex virus type 1 immediate-early regulatory proteins: ICP4 and ICP27 affect the intracellular localization of ICP0.

The results of transient expression assays and studies of viral mutants have shown that three of the five immediate-early proteins of herpes simplex virus type 1 (HSV-1) perform regulatory functions, individually and cooperatively. As part of efforts designed to explore the molecular basis for the functional cooperativity among ICP0, ICP4, and ICP27 in the regulation of HSV gene expression, we have examined the intracellular localization of ICP0 in cells infected with ICP4 and ICP27 null mutant viruses by indirect immunofluorescence. Although ICP0 was localized predominantly to the nuclei of wild-type virus-infected cells, it was found exclusively in the nuclei of ICP27 mutant-infected cells and in both the cytoplasm and nuclei of ICP4 mutant-infected cells, the cytoplasmic component being especially strong. These observations indicate that both ICP4 and ICP27 can affect the intracellular localization of ICP0. Transient expression assays with plasmids that express wild-type and mutant forms of ICP0, ICP4, and ICP27 confirmed that ICP4 promotes and that ICP27 inhibits the nuclear localization of ICP0. These results confirm the observations made for mutant virus-infected cells and indicate that the localization pattern seen in infected cells can be established by these three immediate-early proteins exclusive of other viral proteins. The C-terminal half of ICP27 was shown to be required to achieve its inhibitory effect on the nuclear localization of ICP0. The region of ICP0 responsive to ICP27 was mapped to the C terminus of the molecule between amino acid residues 720 and 769. In addition, the concentration of ICP27 was shown to have a significant effect on the intracellular localization of ICP0. Because the major regulatory activities of ICP0, ICP4, and ICP27 are expressed in the nucleus, the ability of these three proteins collectively to determine their own localization patterns within cells adds a new dimension to the complex process of viral gene regulation in HSV.     

Deletion of the herpes simplex virus VP22-encoding gene (UL49) alters the expression, localization, and virion incorporation of ICP0

The role of the herpes simplex virus tegument protein VP22 is not yet known. Here we describe the characterization of a virus in which the entire VP22 open reading frame has been deleted. We show that VP22 is not essential for virus growth but that virus lacking VP22 (Delta22) displays a cell-specific replication defect in epithelial MDBK cells. Virus particles assembled in the absence of VP22 show few obvious differences to wild-type (WT) particles, except for a moderate reduction in glycoproteins gD and gB. In addition, the Delta22 virus exhibits a general delay in the initiation of virus protein synthesis, but this is not due to a glycoprotein-related defect in virus entry. Intriguingly, however, the absence of VP22 has an obvious effect on the intracellular level of the immediate-early (IE) protein ICP0. Moreover, following translocation from the nucleus to the cytoplasm, ICP0 is unable to localize to the characteristic cytoplasmic sites observed in a WT infection. We demonstrate that, in WT-infected cells, VP22 and ICP0 are concentrated in the same cytoplasmic sites. Furthermore, we show that, while ICP0 and ICP4 are components of WT extracellular virions, the altered localization of ICP0 in the cytoplasm of Delta22-infected cells correlates with an absence of both ICP0 and ICP4 from Delta22 virions. Hence, while a role has not yet been defined for virion IE proteins in virus infection, our results suggest that their incorporation is a specific event requiring the tegument protein VP22. This report provides the first direct evidence that VP22 influences virus assembly.     

Overexpression of the herpes simplex virus type 1 immediate-early regulatory protein, ICP27, is responsible for the aberrant localization of ICP0 and mutant forms of ICP4 in ICP4 mutant virus-infected cells.

ICP0 and ICP4 are immediate-early regulatory proteins of herpes simplex virus type 1. Previous studies by Knipe and Smith demonstrated that these two proteins are characteristically observed in the nuclei of wild-type virus-infected cells but predominantly in the cytoplasms of cells infected with several ICP4 temperature-sensitive (ts) mutant viruses at the nonpermissive temperature (NPT) (D. M. Knipe and J. L. Smith, Mol. Cell. Biol. 6:2371-2381, 1986). Consistent with this observation, it has been shown previously that ICP0 is present predominantly in the cytoplasms of cells infected with an ICP4 null mutant virus (n12) at high multiplicities of infection and that the level of ICP27, a third viral regulatory protein, plays an important role in determining the intracellular localization of ICP0 (Z. Zhu, W. Cai, and P. A. Schaffer, J. Virol. 68:3027-3040, 1994). To address whether the cytoplasmic localization of ICP0 is a common feature of cells infected with all ICP4 mutant viruses or whether mutant ICP4 polypeptides, together with ICP27, determine the intracellular localization of ICP0, we used double-staining immunofluorescence tests to examine the intracellular staining patterns of ICP0 and ICP4 in cells infected with an extensive series of ICP4 mutant viruses. In these tests, compared with the localization pattern of ICP0 in wild-type virus-infected cells, more ICP0 was detected in the cytoplasms of cells infected with all ICP4 mutants tested at high multiplicities of infection. Each of the mutant forms of ICP4 exhibiting predominantly cytoplasmic staining contains both the nuclear localization signal and the previously mapped ICP27-responsive region (Z. Zhu and P. A. Schaffer, J. Virol. 69:49-59, 1995). No correlation between the intracellular staining patterns of ICP0 and mutant forms of ICP4 was demonstrated, suggesting that mutant ICP4 polypeptides per se are not responsible for retention of ICP0 in the cytoplasm. This observation was confirmed in studies of cells cotransfected with plasmids expressing ICP0 and mutant forms of ICP4, in which the staining pattern of ICP0 was not changed in the presence of mutant ICP4 proteins. Studies of cells infected at low multiplicities with a variety of ICP4 ts mutant viruses at the NPT showed that both ICP0 and ts forms of ICP4 were localized predominantly within the nucleus. These observations are a further indication that the aberrant localization of the ts forms of ICP4 at the NPT is not a direct result of specific mutations in the ICP4 gene. In the final series of tests, the localization of ICP0 in cells infected with a double-mutant virus unable to express either ICP4 or ICP27 was examined. In these tests, ICP0 was detected exclusively in the nuclei of Vero cells but in both the nuclei and the cytoplasms of ICP27-expressing cells infected with the double mutant. These results demonstrate that ICP27, rather than the absence of functional ICP4, is responsible for the cytoplasmic localization of ICP0 in ICP4 mutant virus-infected cells. Taken together, these findings demonstrate that the aberrant localization of ICP0 and certain mutant forms of ICP4 in cells infected with ICP4 mutant viruses is mediated by high levels of ICP27 resulting from the inability of mutant forms of ICP4 to repress the expression of ICP27.     

A Network of Protein Interactions around the Herpes Simplex Virus Tegument Protein VP22

Assembly of the herpesvirus tegument is poorly understood but is believed to involve interactions between outer tegument proteins and the cytoplasmic domains of envelope glycoproteins. Here, we present the detailed characterization of a multicomponent glycoprotein-tegument complex found in herpes simplex virus 1 (HSV-1)-infected cells. We demonstrate that the tegument protein VP22 bridges a complex between glycoprotein E (gE) and glycoprotein M (gM). Glycoprotein I (gI), the known binding partner of gE, is also recruited into this gE-VP22-gM complex but is not required for its formation. Exclusion of the glycoproteins gB and gD and VP22''s major binding partner VP16 demonstrates that recruitment of virion components into this complex is highly selective. The immediate-early protein ICP0, which requires VP22 for packaging into the virion, is also assembled into this gE-VP22-gM-gI complex in a VP22-dependent fashion. Although subcomplexes containing VP22 and ICP0 can be formed when either gE or gM are absent, optimal complex formation requires both glycoproteins. Furthermore, and in line with complex formation, neither of these glycoproteins is individually required for VP22 or ICP0 packaging into the virion, but deletion of gE and gM greatly reduces assembly of both VP22 and ICP0. Double deletion of gE and gM also results in small plaque size, reduced virus yield, and defective secondary envelopment, similar to the phenotype previously shown for pseudorabies virus. Hence, we suggest that optimal gE-VP22-gM-gI-ICP0 complex formation correlates with efficient virus morphogenesis and spread. These data give novel insights into the poorly understood process of tegument acquisition.     

Herpes simplex virus ICP27 activation of stress kinases JNK and p38

We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutant tsB7 or with UV-irradiated virus indicated that both JNK and p38 activation required viral gene expression. Cycloheximide reversal or phosphonoacetic acid treatment of wild-type virus-infected cells as well as infection with the ICP4 mutant vi13 indicated that only the immediate-early class of viral proteins were required for SAPK activation. Infection with ICP4, ICP27, or ICP0 mutant viruses indicated that only ICP27 was necessary. Additionally, we determined that in the context of virus infection ICP27 was sufficient for SAPK activation and activation of the p38 targets Mnk1 and MK2 by infecting with mutants deleted for various combinations of immediate-early proteins. Specifically, the d100 (0-/4-) and d103 (4-/22-/47-) mutants activated p38 and JNK, while the d106 (4-/22-/27-/47-) and d107 (4-/27-) mutants did not. Finally, infections with a series of ICP27 mutants demonstrated that the functional domain of ICP27 required for activation was located in the region encompassing amino acids 20 to 65 near the N terminus of the protein and that the C-terminal transactivation activity of ICP27 was not necessary.     

Mutations in the activation region of herpes simplex virus regulatory protein ICP27 can be trans dominant.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein which is required for virus replication. Transfection experiments have demonstrated that ICP27 along with the HSV-1 transactivators ICP4 and ICP0 can positively regulate the expression of some late HSV-1 target plasmids and can negatively regulate the expression of some immediate-early and early target plasmids. We previously showed that mutants defective in the activation of a late target plasmid mapped to the carboxy-terminal half of the protein, whereas mutants defective in the repression of an early target plasmid mapped within the C-terminal 78 amino acids of ICP27 (M. A. Hardwicke, P. J. Vaughan, R. E. Sekulovich, R. O'Conner, and R. M. Sandri-Goldin, J. Virol. 63:4590-4602, 1989). In this study, we cotransfected ICP27 activator and repressor mutants along with wild-type ICP27 plasmid to determine whether these mutants could interfere with the wild-type activities. Mutants which were defective only in the activation function were dominant to the wild-type protein and inhibited the activation of the late target plasmid pVP5-CAT, whereas mutants defective in the repressor function did not inhibit either the activation of pVP5-CAT or the repression of the early target plasmid pTK-CAT. Furthermore, cell lines which stably carried three different activator mutants were impaired in their ability to support the growth of wild-type HSV-1 strain KOS, resulting in virus yields 5- to 40-fold lower than in control cells. The defect in virus replication appeared to stem from a decrease in the expression of HSV-1 late gene products during infection as measured by steady-state mRNA levels and by immunoprecipitation analysis of specific polypeptides. These results indicate that ICP27 activator mutations specifically interfere with the activation function of the protein both in transfection and during infection. Moreover, these results suggest that the repressor region may be important for binding of the polypeptide, since mutations in this region did not interfere with the activities of wild-type ICP27 and therefore presumably could not compete for binding.     

Expression of herpes simplex virus ICP0 inhibits the induction of interferon-stimulated genes by viral infection

The herpes simplex virus type 1 (HSV-1) mutant d109 does not express any of the immediate-early (IE) proteins and persists in cells for a prolonged length of time. As has been shown by Nicholl et al. (J. Gen. Virol. 81:2215-2218, 2000) and Mossman et al. (J. Virol. 75:750-758, 2001) using other mutants defective for IE gene expression, infection with d109 induced the expression of a number of interferon-stimulated genes. Induction of these genes was significantly greater at multiplicities of infection (MOI) of 10 PFU/cell or greater, and the resulting antiviral effect was only seen at MOIs greater than 10 PFU/cell. Using mutants defective for sets of IE genes established that the lack of ICP0 expression was necessary for high levels of interferon-stimulated gene expression in HEL cells. The induction of interferon-stimulated genes by d109 could also be inhibited by infection with an E1-:E3-:E4- adenovirus expressing levels of ICP0 that are comparable to those expressed within the first hour of wild-type virus infection. Lastly, the addition of the proteasome inhibitor MG132 to cells infected with a mutant that expresses ICP0, d106, also resulted in the induction of interferon-stimulated genes. Thus, ICP0 may function through the proteasome very early in HSV infection to inhibit a cellular antiviral response induced by the virion.     

Neurons differentially activate the herpes simplex virus type 1 immediate-early gene ICP0 and ICP27 promoters in transgenic mice

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins are required for the expression of viral early and late proteins. It has been hypothesized that host neuronal proteins regulate expression of HSV-1 IE genes that in turn control viral latency and reactivation. We investigated the ability of neuronal proteins in vivo to activate HSV-1 IE gene promoters (ICP0 and ICP27) and a late gene promoter (gC). Transgenic mice containing IE (ICP0 and ICP27) and late (gC) gene promoters of HSV-1 fused to the Escherichia coli beta-galactosidase coding sequence were generated. Expression of the ICP0 and ICP27 reporter transgenes was present in anatomically distinct subsets of neurons in the absence of viral proteins. The anatomic locations of beta-galactosidase-positive neurons in the brains of ICP0 and ICP27 reporter transgenic mice were similar and included cerebral cortex, lateral septal nucleus, cingulum, hippocampus, thalamus, amygdala, and vestibular nucleus. Trigeminal ganglion neurons were positive for beta-galactosidase in adult ICP0 and ICP27 reporter transgenic mice. The ICP0 reporter transgene was differentially regulated in trigeminal ganglion neurons depending upon age. beta-galactosidase-labeled cells in trigeminal ganglia and cerebral cortex of ICP0 and ICP27 reporter transgenic mice were confirmed as neurons by double labeling with antineurofilament antibody. Nearly all nonneuronal cells in ICP0 and ICP27 reporter transgenic mice and all neuronal and nonneuronal cells in gC reporter transgenic mice were negative for beta-galactosidase labeling in the absence of HSV-1. We conclude that factors in neurons are able to differentially regulate the HSV-1 IE gene promoters (ICP0 and ICP27) in transgenic mice in the absence of viral proteins. These findings are important for understanding the regulation of the latent and reactivated stages of HSV-1 infection in neurons.