Herpes simplex virus type 1 primary envelopment: UL34 protein modification and the US3-UL34 catalytic relationship

The herpes simplex virus type 1 (HSV-1) US3 kinase is likely important for primary envelopment of progeny nucleocapsids since it localizes to the nuclear envelope of infected cells and largely determines the phosphorylation state and localization of the necessary primary envelopment factor, the UL34 protein. In HEp-2 cells, the production of infectious US3 null progeny is delayed and decreased relative to that of the parental strain, HSV-1(F). Furthermore, the US3 kinase affects the morphology of primary envelopment such that in its absence, UL34 protein-containing enveloped virions accumulate within membrane-bound vesicles. These vesicles are most often found along the interior periphery of the nucleus and may be derived from the inner nuclear membrane. Since the US3 and UL34 proteins comprise a kinase-substrate pair, a reasonable hypothesis is that the US3 kinase influences these replication parameters by direct phosphorylation of the UL34 protein. For this report, recombinant viruses were constructed to determine the significance of UL34 protein phosphorylation and US3 catalytic activity on UL34 protein localization, single-step growth, and envelopment morphology in both HEp-2 and Vero cells. The data presented suggest that the significance of UL34 phosphorylation is cell type dependent and that efficient viral morphogenesis requires US3-mediated phosphorylation of an infected cell protein other than UL34.     

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 of proteins phosphorylated directly by the Us3 protein kinase encoded by herpes simplex virus 1

We have developed a system to analyze the specific protein kinase activity of herpes simplex virus 1 Us3 in vitro and shown that Us3 directly phosphorylates viral proteins UL34, ICP22, and Us9 and the cellular protein Bad, previously reported to be putative substrates. Using this system, we determined the phosphorylation sites of UL34 and identified UL31 as a previously unreported, novel substrate of Us3. This system will be useful for further identification of Us3 substrates and their phosphorylation sites, clarification of the role of Us3 in viral replication, and identification of additional Us3 function(s).     

Identification of proteins directly phosphorylated by UL13 protein kinase from herpes simplex virus 1

Herpes simplex virus 1 (HSV-1) UL13 is a viral protein kinase that regulates optimal viral replication in cell cultures. Identification of substrates of protein kinases is a crucial step to elucidate the mechanism by which they function. Using our developed system to analyze the specific protein kinase activity of UL13, we have shown that UL13 protein kinase directly phosphorylates the viral proteins ICP22 and UL49 previously reported to be putative substrates. We also identified UL41 as a previously unreported and novel substrate of UL13. These data will serve as a basis to clarify the mechanism by which UL13 influences viral replication.     

Cell-type-specific tyrosine phosphorylation of the herpes simplex virus tegument protein VP11/12 encoded by gene UL46

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play key roles in limiting herpesvirus infections; consequently, many herpesviruses, including herpes simplex virus (HSV), have evolved diverse strategies to evade and/or disarm these killer lymphocytes. Previous studies have shown that CTL and NK cells are functionally inactivated following contact with HSV-infected fibroblasts. During studies of the mechanisms involved, we discovered that HSV-inactivated NK-92 NK cells and Jurkat T cells contain a strikingly prominent, novel, ca. 90-kDa tyrosine-phosphorylated protein that we identified as the HSV tegument protein VP11/12. Inasmuch as VP11/12 produced in fibroblasts and epithelial cells is not obviously tyrosine phosphorylated, these data suggested that VP11/12 serves as the substrate of a cell-type-specific protein tyrosine kinase. Consistent with this hypothesis, VP11/12 was also tyrosine phosphorylated in B lymphocytes, and this modification was severely reduced in Jurkat T cells lacking the lymphocyte-specific Src family kinase Lck. These findings demonstrate that HSV tegument proteins can be differentially modified depending on the cell type infected. Our data also raise the possibility that VP11/12 may modulate one or more lymphocyte-specific signaling pathways or serve another lymphocyte-specific function. However, HSV type 1 mutants lacking the UL46 gene retained the ability to block signaling through the T-cell receptor in Jurkat cells and remained competent to functionally inactivate the NK-92 NK cell line, indicating that VP11/12 is not essential for lymphocyte inactivation. Further studies are therefore required to determine the biological function of tyrosine-phosphorylated VP11/12.     

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.     

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.     

Phosphorylation of cytokeratin 17 by herpes simplex virus type 2 US3 protein kinase

We previously reported the establishment of an HEp2 cell line which expresses the US3 protein kinase (PK) of herpes simplex virus type 2 (HSV-2) upon induction with IPTG. Here we report that expression, phosphorylation and ubiquitination of cytokeratin 17 (CK17) are enhanced in US3-expressing HEp2 cells. In vitro kinase and co-immunoprecipitation assays provided evidence that US3 PK directly phosphorylates CK17. Expression of US3 PK caused a significant decrease in filamentous staining of CK17, suggesting that phosphorylation of CK17 by US3 PK causes a disruption of intermediate filaments. Our observations suggest a role for US3 in the regulation of CKs and intermediate filaments in cells. Moreover, we found that infection of a keratinocyte-derived cell line, A431, with a US3-deficient virus, results in cytopathic effects that are morphologically distinct from those induced by wild-type and revertant viruses, suggesting that US3 PK may be important for interaction between HSV-2 and peripheral epithelial cells.     

Antiapoptotic activity of herpes simplex virus type 2: the role of US3 protein kinase gene

In order to determine the ability of herpes simplex virus type 2 (HSV-2) to suppress apoptosis, we examined the effect of HSV-2 infection on apoptosis induced in HEp-2 cells by treatment with 1 M sorbitol. Although a wild-type strain of HSV-2 induced apoptosis in a significant fraction of the infected cells, HSV-2 could suppress sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that HSV-2, like HSV-1, has an antiapoptosis gene. Characterization of the cells infected with a US3-deletion mutant of HSV-2 revealed the necessity of a US3 gene in the antiapoptotic activity of this virus.     

Herpes simplex virus 1-encoded protein kinase UL13 phosphorylates viral Us3 protein kinase and regulates nuclear localization of viral envelopment factors UL34 and UL31

UL13 and Us3 are protein kinases encoded by herpes simplex virus 1. We report here that Us3 is a physiological substrate for UL13 in infected cells, based on the following observations. (i) The electrophoretic mobility, in denaturing gels, of Us3 isoforms from Vero cells infected with wild-type virus was slower than that of isoforms from cells infected with a UL13 deletion mutant virus (DeltaUL13). After treatment with phosphatase, the electrophoretic mobility of the Us3 isoforms from cells infected with wild-type virus changed, with one isoform migrating as fast as one of the Us3 isoforms from DeltaUL13-infected cells. (ii) A recombinant protein containing a domain of Us3 was phosphorylated by UL13 in vitro. (iii) The phenotype of DeltaUL13 resembles that of a recombinant virus lacking the Us3 gene (DeltaUs3) with respect to localization of the viral envelopment factors UL34 and UL31, whose localization has been shown to be regulated by Us3. UL34 and UL31 are localized in a smooth pattern throughout the nuclei of cells infected with wild-type virus, whereas their localization in DeltaUL13- and DeltaUs3-infected cells appeared as nuclear punctate patterns. These results indicate that UL13 phosphorylates Us3 in infected cells and regulates UL34 and UL31 localization, either by phosphorylating Us3 or by a Us3-independent mechanism.     

Effects of charged cluster mutations on the function of herpes simplex virus type 1 UL34 protein

Herpes simplex virus type 1 (HSV-1) is a DNA virus that acquires an envelope by budding into the inner nuclear membrane of an infected cell. Recombinant HSV-1 lacking the U(L)34 gene cannot undergo this event. U(L)34 and U(L)31, another viral protein, colocalize in an infected cell and are necessary and sufficient to target both proteins to the inner nuclear envelope. In order to define and characterize sequences of U(L)34 that are necessary for primary envelopment to occur, a library of 19 U(L)34 charged cluster mutants and a truncation mutant lacking the putative transmembrane domain (DeltaTM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Seven of the mutants failed to complement a U(L)34-null virus. The remainder of the mutants complemented at or near wild-type U(L)34 levels. Failure of a mutant protein to function might be the result of incorrect subcellular localization. To address this possibility, confocal microscopy was used to determine the localization of the U(L)34 protein in charged cluster mutants and DeltaTM. In transfection-infection experiments, all of the functional U(L)34 mutants and four of the six noncomplementing mutants localized to the inner nuclear envelope in a manner indistinguishable from that of wild-type U(L)34. All of the noncomplementing U(L)34 mutants mediated proper localization of U(L)31. Charged clusters critical for U(L)34 function are dispersed throughout the protein sequence and do not correlate well with highly conserved regions of the protein. These data suggest that U(L)34 has at least one function in addition to mediating proper localization of U(L)31 in infected cells and provide further support for the role of U(L)34 in mediating proper localization of U(L)31 in infected cells.     

The product of the UL31 gene of herpes simplex virus 1 is a nuclear phosphoprotein which partitions with the nuclear matrix.

The nucleotide sequence of the UL31 open reading frame is predicted to encode a basic protein with a hydrophilic amino terminus and a nuclear localization signal. To identify its gene product, we constructed a viral genome in which the thymidine kinase gene was inserted between the UL31 and UL32 open reading frames. The thymidine kinase gene was then deleted, and in the process, the 5' terminus of the UL31 open reading frame was replaced with a 64-bp sequence in frame with the complete, authentic sequence of the UL31 open reading frame. The inserted sequence encoded a hydrophilic epitope derived from glycoprotein B of human cytomegalovirus and for which a monoclonal antibody is available. We report that in infected cells, the tagged protein localized in and was dispersed throughout the nucleus. Nuclear fractionation studies revealed that the UL31 protein partitions with the nuclear matrix. The protein is phosphorylated in infected cells maintained in medium containing 32Pi.     

The herpes simplex virus 1 US3 protein kinase blocks caspase-dependent double cleavage and activation of the proapoptotic protein BAD

An earlier report showed that the U(S)3 protein kinase blocked the apoptosis induced by the herpes simplex virus 1 (HSV-1) d120 mutant at a premitochondrial stage. Further studies revealed that the kinase also blocks programmed cell death induced by the proapoptotic protein BAD. Here we report the effects of the U(S)3 protein kinase on the function and state of a murine BAD protein. Specifically, (i) in uninfected cells, BAD was processed by at least two proteolytic cleavages that were blocked by a general caspase inhibitor. The untreated transduced cells expressed elevated caspase 3 activity. (ii) In cells cotransduced with the U(S)3 protein kinase, the BAD protein was not cleaved and the caspase 3 activity was not elevated. (iii) Inasmuch as the U(S)3 protein kinase blocked the proapoptotic activity and cleavage of a mutant (BAD3S/A) in which the codons for the regulatory serines at positions 112, 136, and 155 were each replaced with alanine codons, the U(S)3 protein kinase does not act by phosphorylation of these sites nor was the phosphorylation of these sites required for the antiapoptotic function of the U(S)3 protein kinase. (iv) The U(S)3 protein kinase did not enable the binding of the BAD3S/A mutant to the antiapoptotic proteins 14-3-3. Finally, (v) whereas cleavage of BAD at ASP56 and ASP61 has been reported and results in the generation of a more effective proapoptotic protein with an M(r) of 15,000, in this report we also show the existence of a second caspase-dependent cleavage site most likely at the ASP156 that is predicted to inactivate the proapoptotic activity of BAD. We conclude that the primary effect of U(S)3 was to block the caspases that cleave BAD at either residue 56 or 61 predicted to render the protein more proapoptotic or at residue 156, which would inactivate the protein.     

Functional analysis of the herpes simplex virus UL42 protein.

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

The pseudorabies virus UL28 protein enters the nucleus after coexpression with the herpes simplex virus UL15 protein.

Herpesvirus DNA is packaged into capsids in the nuclei of infected cells in a process requiring at least six viral proteins. Of the proteins required for encapsidation of viral DNA, UL15 and UL28 are the most conserved among herpes simplex virus type 1 (HSV), varicella-zoster virus, and equine herpesvirus 1. The subcellular distribution of the pseudorabies virus (PRV) UL28 protein was examined by in situ immunofluorescence. UL28 was present in the nuclei of infected cells; however, UL28 was limited to the cytoplasm in the absence of other viral proteins. When cells expressing variant forms of UL28 were infected with a PRV UL28-null mutant, UL28 entered the nucleus, provided the carboxyl-terminal 155 amino acids were present. Additionally, PRV UL28 entered the nucleus in cells infected with HSV. Two HSV packaging proteins were tested for the ability to affect the subcellular distribution of UL28. Coexpression of HSV UL15 enabled PRV UL28 to enter the nucleus in a manner that required the carboxyl-terminal 155 amino acids of UL28. Coexpression of HSV UL25 did not affect the distribution of UL28. We propose that an interaction between UL15 and UL28 facilitates the transport of a UL15-UL28 complex to the infected-cell nucleus.     

The positively charged surface of herpes simplex virus UL42 mediates DNA binding

Herpes simplex virus DNA polymerase is a heterodimer composed of UL30, a catalytic subunit, and UL42, a processivity subunit. Mutations that decrease DNA binding by UL42 decrease long chain DNA synthesis by the polymerase. The crystal structure of UL42 bound to the C terminus of UL30 revealed an extensive positively charged surface ("back face"). We tested two hypotheses, 1) the C terminus of UL30 affects DNA binding and 2) the positively charged back face mediates DNA binding. Addressing the first hypothesis, we found that the presence of a peptide corresponding to the UL30 C terminus did not result in altered binding of UL42 to DNA. Addressing the second hypothesis, previous work showed that substitution of four conserved arginine residues on the basic face with alanines resulted in decreased DNA affinity. We tested the affinities for DNA and the stimulation of long chain DNA synthesis of mutants in which the four conserved arginine residues were substituted individually or together with lysines and also a mutant in which a conserved glutamine residue was substituted with an arginine to increase positive charge on the back face. We also engineered cysteines onto this surface to permit disulfide cross-linking studies. Last, we assayed the effects of ionic strength on DNA binding by UL42 to estimate the number of ions released upon binding. Our results taken together strongly suggest that the basic back face of UL42 contacts DNA and that positive charge on this surface is important for this interaction.     

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.     

Identification of an essential domain in the herpes simplex virus 1 UL34 protein that is necessary and sufficient to interact with UL31 protein

Previous results have indicated that the herpes simplex virus 1 UL31 and UL34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione S-transferase pull-down assays indicated that amino acids 137 to 181 of the UL34 protein are sufficient to mediate an interaction with the UL31 protein. A recombinant virus (v3480) lacking UL34 codons 138 to 181 was constructed. Similar to a UL34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A UL34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of UL31 protein in cells infected with a UL34 null virus, the UL31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the UL34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a UL31 null virus. In transient expression assays, the interaction between UL34 amino acids 137 to 181 and the UL31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the UL31 protein. These data indicate that amino acids 137 to 181 of the UL34 protein are (i) sufficient to mediate interactions with the UL31 protein in vitro and in vivo, (ii) necessary for the colocalization of UL31 and UL34 in infected cells, and (iii) essential for normal viral replication.