Herpes simplex virus type 1 infection induces activation and recruitment of protein kinase C to the nuclear membrane and increased phosphorylation of lamin B

We report that herpes simplex virus type 1 (HSV-1) infection leads to the recruitment of protein kinase C (PKC) to the nuclear rim. In HEp-2 cells, PKC recruitment to the nuclear rim was initiated between 8 h and 12 h postinfection. PKCdelta, a proapoptotic kinase, was completely recruited to the nuclear rim upon infection with HSV-1. PKCalpha was less dramatically relocalized mostly at the nuclear rim upon infection, although some PKCalpha remained in the cytoplasm. PKCzeta-specific immunofluorescence was not significantly relocated to the nuclear rim. The UL34 and UL31 proteins, as well as their association, were each required for PKC recruitment to the nuclear rim. The HSV-1 US3 protein product, a kinase which regulates the phosphorylation state and localization of UL34, was not required for PKC recruitment to the nuclear rim; however, it was required for proper localization along the nuclear rim, as PKC appeared unevenly distributed along the nuclear rim of cells infected with US3 null and kinase-dead mutants. HSV-1 infection induced the phosphorylation of both lamin B and PKC. Elevated lamin B phosphorylation in HSV-1-infected cells was partially reduced by inhibitors of PKC. The data suggest a model in which kinases that normally disassemble the nuclear lamina during apoptosis are recruited to the nuclear membrane through functions requiring UL31 and UL34. We hypothesize that the recruitment of PKC functions to phosphorylate lamin B to help modify the nuclear lamina and promote budding of nucleocapsids at the inner nuclear membrane.     

Emerin is hyperphosphorylated and redistributed in herpes simplex virus type 1-infected cells in a manner dependent on both UL34 and US3

Cells infected with wild-type herpes simplex virus type 1 (HSV-1) show disruption of the organization of the nuclear lamina that underlies the nuclear envelope. This disruption is reflected in changes in the localization and phosphorylation of lamin proteins. Here, we show that HSV-1 infection causes relocalization of the LEM domain protein emerin. In cells infected with wild-type virus, emerin becomes more mobile in the nuclear membrane, and in cells infected with viruses that fail to express UL34 protein (pUL34) and US3 protein (pUS3), emerin no longer colocalizes with lamins, suggesting that infection causes a loss of connection between emerin and the lamina. Infection causes hyperphosphorylation of emerin in a manner dependent upon both pUL34 and pUS3. Some emerin hyperphosphorylation can be inhibited by the protein kinase Cdelta (PKCdelta) inhibitor rottlerin. Emerin and pUL34 interact physically, as shown by pull-down and coimmunoprecipitation assays. Emerin expression is not, however, necessary for infection, since virus growth is not impaired in cells derived from emerin-null transgenic mice. The results suggest a model in which pUS3 and PKCdelta that has been recruited by pUL34 hyperphosphorylate emerin, leading to disruption of its connections with lamin proteins and contributing to the disruption of the nuclear lamina. Changes in emerin localization, nuclear shape, and lamin organization characteristic of cells infected with wild-type HSV-1 also occur in cells infected with recombinant virus that does not make viral capsids, suggesting that these changes occur independently of capsid envelopment.     

Recruitment of cellular recombination and repair proteins to sites of herpes simplex virus type 1 DNA replication is dependent on the composition of viral proteins within prereplicative sites and correlates with the induction of the DNA damage response

Herpes simplex virus type 1 (HSV-1) DNA replication is associated with nuclear domains called ND10, which contain host recombination proteins such as RPA, RAD51, and NBS1 and participate in the cell's response to DNA damage. The stages of HSV-1 infection have been described previously. Infected cells at stage IIIa are observed after the initial disruption of ND10 and display nuclear foci, or prereplicative sites, containing the viral single-stranded-DNA-binding protein (UL29), the origin-binding protein (UL9), and the heterotrimeric helicase-primase. At stage IIIb, the viral polymerase, its processivity factor, and the ND10, protein PML, are also recruited to these sites. In this work, RPA, RAD51, and NBS1 were observed predominantly in stage IIIb but not stage IIIa prereplicative sites, suggesting that the efficient recruitment of these recombination proteins is dependent on the presence of the viral polymerase and other replication proteins within these sites. On the other hand, Ku86 was not found in any of the precursors to replication compartments, suggesting that it is excluded from the early stages of HSV-1 replication. Western blot analysis showed that RPA and NBS1 were (hyper)phosphorylated during infection, indicating that infection induces the host response to DNA damage. Finally, RPA, RAD51, and NBS1 were found to be associated with UL29 foci observed in transfected cells expressing UL29 and the helicase-primase heterotrimer and containing intact ND10. The ability to recruit recombination and repair proteins to various subassemblies of viral replication proteins thus appears to depend on several factors, including the presence of the viral polymerase and/or UL9 within prereplicative sites and the integrity of ND10.     

A mutation in the human herpes simplex virus type 1 UL52 zinc finger motif results in defective primase activity but can recruit viral polymerase and support viral replication efficiently

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase/primase complex consisting of UL5, UL8, and UL52. UL5 contains conserved helicase motifs, while UL52 contains conserved primase motifs, including a zinc finger motif. Although HSV-1 and HSV-2 UL52s contain a leucine residue at position 986, most other herpesvirus primase homologues contain a phenylalanine at this position. We constructed an HSV-1 UL52 L986F mutation and found that it can complement a UL52 null virus more efficiently than the wild type (WT). We thus predicted that the UL5/8/52 complex containing the L986F mutation might possess increased primase activity; however, it exhibited only 25% of the WT level of primase activity. Interestingly, the mutant complex displayed elevated levels of DNA binding and single-stranded DNA-dependent ATPase and helicase activities. This result confirms a complex interdependence between the helicase and primase subunits. We previously showed that primase-defective mutants failed to recruit the polymerase catalytic subunit UL30 to prereplicative sites, suggesting that an active primase, or primer synthesis, is required for polymerase recruitment. Although L986F exhibits decreased primase activity, it can support efficient replication and recruit UL30 efficiently to replication compartments, indicating that a partially active primase is capable of recruiting polymerase. Extraction with detergents prior to fixation can extract nucleosolic proteins but not proteins bound to chromatin or the nuclear matrix. We showed that UL30 was extracted from replication compartments while UL42 remained bound, suggesting that UL30 may be tethered to the replication fork by protein-protein interactions.     

Interactions of herpes simplex virus type 1 with ND10 and recruitment of PML to replication compartments

Many of the events required for productive herpes simplex virus type 1 (HSV-1) infection occur within globular nuclear domains called replication compartments, whose formation appears to depend on interactions with cellular nuclear domains 10 (ND10). We have previously demonstrated that the formation of HSV-1 replication compartments involves progression through several stages, including the disruption of intact ND10 (stage I to stage II) and the formation of PML-associated prereplicative sites (stage III) and replication compartments (stage IV) (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). In this paper, we show that some, but not all, PML isoforms are recruited to stage III foci and replication compartments. Genetic experiments showed that the recruitment of PML isoforms to stage III prereplicative sites and replication compartments requires the localization of the HSV-1 polymerase protein (UL30) to these foci but does not require polymerase catalytic activity. We also examined the stages of viral infection under conditions affecting ND10 integrity. Treatment with factors that increase the stability of ND10, arsenic trioxide and the proteasome inhibitor MG132, inhibited viral disruption of ND10, formation of replication compartments, and production of progeny virus. These results strengthen the previously described correlation between ND10 disruption and productive viral infection.     

p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress

As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.     

Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids

Interaction between pUL34 and pUL31 is essential for targeting both proteins to the inner nuclear membrane (INM). Sequences mediating the targeting interaction have been mapped by others with both proteins. We have previously reported identification of charge cluster mutants of herpes simplex virus type 1 UL34 that localize properly to the inner nuclear membrane, indicating interaction with UL31, but fail to complement a UL34 deletion. We have characterized one mutation (CL04) that alters a charge cluster near the N terminus of pUL34 and observed the following. (i) The CL04 mutant has a dominant-negative effect on pUL34 function, indicating disruption of some critical interaction. (ii) In infections with CL04 pUL34, capsids accumulate in close association with the INM, but no perinuclear enveloped viruses, cytoplasmic capsids, or virions or cell surface virions were observed, suggesting that CL04 UL34 does not support INM curvature around the capsid. (iii) Passage of UL34-null virus on a stable cell line that expresses CL04 resulted in selection of extragenic suppressor mutants that grew efficiently using the mutant pUL34. (iv) All extragenic suppressors contained an R229→L mutation in pUL31 that was sufficient to suppress the CL04 phenotype. (v) Immunolocalization and coimmunoprecipitation experiments with truncated forms of pUL34 and pUL31 confirm that N-terminal sequences of pUL34 and a C-terminal domain of pUL31 mediate interaction but not nuclear membrane targeting. pUL34 and pUL31 may make two essential interactions—one for the targeting of the complex to the nuclear envelope and another for nuclear membrane curvature around capsids.Egress of herpesvirus capsids from the nucleus occurs by envelopment of capsids at the inner nuclear membrane (INM) and is followed by de-envelopment at the outer nuclear membrane (ONM). This process can be broken down into a pathway of discrete steps that begin with recruitment of the viral envelopment apparatus to the INM. Herpes simplex virus type 1 (HSV-1) UL34 and UL31 and their homologs in other herpesviruses are required for efficient envelopment at the INM (7, 13, 22, 23, 29). HSV-1 pUL31 and pUL34 are targeted specifically to the INM by a mechanism that requires their interaction with each other (27, 28), and this mutual dependence is a conserved feature of herpesvirus envelopment (9, 14, 27, 28, 32, 33, 39). Localization of these two proteins at the INM results in the recruitment of other proteins, including protein kinase C delta and pUS3, to the nuclear membrane (22, 24, 30). The sequences in HSV-1 pUL34 that mediate interaction with UL31 and that lead to nuclear envelope targeting were mapped to amino acids (aa) 137 to 181 (16). The sequences in the murine cytomegalovirus (MCMV) homolog of UL31, M53, that mediate the nuclear envelope targeting interaction with the UL34 homolog, M50, were mapped to the N-terminal third of the protein in the first of four conserved regions (17), and Schnee et al. subsequently showed that this same region of pUL31 homologs from other families of herpesviruses mediates interaction with the corresponding pUL34 homologs (33).After the targeting of the pUL34/pUL31 complex to the INM, subsequent steps in nuclear egress include, it is thought, (i) local disruption of the nuclear lamina to allow capsid access to the INM, (ii) recognition and docking of capsids by the envelopment apparatus at the INM, (iii) curvature of the inner and outer nuclear membranes around the capsid, (iv) scission of the INM to create an enveloped virion in the space between the INM and ONM, (v) fusion of the virion envelope with the outer nuclear membrane, and (vi) capsid release into the cytoplasm.At least some of the viral and cellular factors critical for nuclear lamina disruption and for de-envelopment fusion have been identified. pUL34, pUL31, and pUS3 of HSV-1 have all been implicated in changes in localization, interaction, and phosphorylation of nuclear lamina components, including lamins A/C and B and the lamina-associated protein, emerin (3, 15, 19, 20, 24, 26, 34, 35). pUS3, pUL31, and glycoproteins B and H have been implicated in de-envelopment of primary virions at the ONM (8, 21, 28, 30, 38).pUL34 and pUL31 are thought to be involved in steps between lamina disruption and de-envelopment, but genetic evidence in infected cells has so far been lacking. Klupp et al. have shown that overexpression of alphaherpesvirus pUL31 and pUL34 in the absence of other viral proteins can induce formation of small vesicles derived from the INM, suggesting a role for these two proteins in membrane curvature around the capsid (12). Tight membrane curvature is an energetically unfavorable event and is thought to be accomplished by coupling curvature to energetically favorable interactions between membrane-bound proteins or protein complexes (reviewed in reference 40). The data of Klupp et al. suggest the possibility that upon recognition of a capsid, pUL31 and pUL34 may interact in a way that induces tight curvature of the INM. Here we present data in support of this hypothesis, showing that a specific point mutation in UL34 induces accumulation of docked capsids at the INM, extragenic suppression of the mutant phenotype is associated with a mutation in UL31, and pUL31 and pUL34 can interact via sequences that are not involved in their INM targeting interaction.We previously published a characterization of a library of 19 charge cluster mutants of pUL34. In each of these mutants, one charge cluster (defined as a group of five consecutive amino acids in which two or more of the residues have charged side chains) was mutated such that the charged residues were replaced by alanine. Six of the 19 charge cluster mutants tested failed to complement replication of UL34-null virus, indicating that they disrupt essential functions of pUL34. Interestingly, five of the six noncomplementing mutants were synthesized at levels comparable to that of wild-type UL34 and localized normally to the nuclear envelope, suggesting that they were unimpaired in their ability to make a nuclear envelope targeting interaction with UL31. In order to identify essential functions of pUL34 downstream of nuclear envelope targeting, we have undertaken a detailed study of the behavior and interactions of these mutants.     

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.     

pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.     

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase δ was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.Adeno-associated virus (AAV) is a human parvovirus that is currently used as a gene transfer vector (14). AAV particles consist of a small icosahedral capsid protecting a single 4.7-kb single-stranded DNA (ssDNA) genome with two open reading frames, rep and cap, surrounded by inverted terminal repeats (ITRs). The ITRs are the only sequences required in cis for genome replication and packaging. The rep gene encodes four nonstructural Rep proteins: Rep78, -68, -52, and -40. The two larger isoforms, Rep78 and -68, have origin binding, helicase, and site-specific endonuclease activities and are involved in AAV gene expression and genome processing, including replication and site-specific integration (39). The two smaller Rep isoforms are not required for AAV DNA replication but are involved in the control of viral gene expression and packaging of viral DNA (30).When wild-type (wt) AAV infects a cell in the absence of a helper virus, it enters latency. Latent AAV genomes persist in cells either as episomes or as integrated genomes, preferentially at a specific locus (named AAVS1) on human chromosome 19. In most instances, no detectable viral gene expression or genome replication occurs unless the cell is co- or superinfected by a helper virus, such as adenovirus, herpes simplex virus type 1 (HSV-1), or HSV-2. Under these conditions, AAV replication and assembly take place in large intranuclear domains called replication compartments (RCs) that frequently colocalize with replication domains formed by the helper virus itself (81). The viral genome replicates by leading-strand synthesis and generates new ssDNA molecules by a strand displacement mechanism that occurs after strand- and site-specific cleavage of viral DNA by Rep78/68 within the ITRs (39).Studies conducted on the relationship between AAV and its helper viruses are important not only to identify helper activities that can be used to produce recombinant AAV vectors but also to understand how AAV adapts its replication strategy to the helper virus and to the nuclear environment in general. Adenovirus helper functions have historically been the first and most extensively studied functions. These studies have shown that adenovirus helps AAV by stimulating viral gene expression and by enhancing AAV genome replication, mostly indirectly (19). Indeed, early studies showed that the adenovirus polymerase (E2b) is dispensable for AAV replication (8) and that the viral DNA-binding protein (DBP), the product of the E2a gene, is able to modestly enhance the processivity of AAV genome replication in vitro (77). More recently, the adenovirus proteins E1b55k and E4orf6 were shown to stimulate AAV genome replication by degrading the cellular Mre11/Rad50/Nbs1 (MRN) complex that restricts AAV genome replication during adenovirus coinfection (32). The concept that AAV genome replication can rely mostly, if not uniquely, on direct help from cellular factors was further strengthened by the demonstration that purified proteins such as replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), minichromosome maintenance (MCM) proteins, and DNA polymerase δ (Pol δ) were sufficient to replicate the AAV genome in vitro in the presence of Rep (40-41, 43). The involvement of these cellular proteins during AAV genome replication was also confirmed by the proteomic analysis of factors associated with Rep proteins during adenovirus-induced AAV replication (42).Interestingly, studies conducted on HSV-1 helper activities suggest that the strategy of AAV replication may vary depending on the helper virus. Indeed, previous studies showed that the HSV-1 helicase-primase (HP) complex (UL5/8/52) and DBP (ICP8) could replicate transfected AAV-2 plasmids (80) and that the helicase activity, but not primase activity, of the HP complex was required for this effect (62, 66). More recently, a comprehensive study of HSV-1 helper activities demonstrated that the HSV-1 immediate-early proteins ICP0, ICP4, and ICP22 could stimulate rep gene expression, probably by diminishing intrinsic antiviral effects (1, 18). In addition, the HSV-1 DNA polymerase encoded by UL30, along with its associated processivity factor (UL42), although not strictly required, was demonstrated to significantly increase AAV replication levels induced in the presence of the HP complex and ICP8. Interestingly, the HSV-1 HP complex, DBP, and polymerase were also shown to be sufficient to replicate AAV DNA in vitro in the presence of Rep proteins without any cellular protein (78). Altogether, these observations indicate that in the context of an HSV-1 coinfection, AAV relies extensively on viral activities provided by the helper that directly participate in AAV genome replication.To further elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis to identify the cellular and HSV-1 factors associated with Rep proteins and, consequently, potentially recruited within AAV RCs. To analyze Rep-associated proteins in the presence and absence of HSV-1 DNA replication, this analysis was performed using wt HSV-1 and an HSV-1 mutant in which the DNA polymerase encoded by the UL30 gene is absent (HSVΔUL30). This study resulted in the identification of approximately 60 cellular proteins, among which the largest functional categories corresponded to factors involved in DNA and RNA metabolism. Immunofluorescence analyses confirmed that in the presence of HSV-1, a basal set of cellular DNA replication enzymes, including RPA, RFC, and PCNA, was recruited within AAV RCs, with the exception of the MCM helicases. The cellular DNA polymerases, in particular Pol δ, were not identified by this analysis but subsequently were shown to be recruited in AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, our results indicate that AAV replication induced by HSV-1 is associated with the recruitment of DNA repair factors, including components of the MRN complex, the Ku proteins, PARP-1, and factors of the mismatch repair (MMR) pathway. Finally, several HSV-1 proteins, most notably the UL12 protein, were also identified within AAV RCs. Our analyses confirmed the association between UL12 and Rep and demonstrated for the first time that this viral exonuclease plays a critical role during AAV replication by enhancing the formation of discrete AAV replicative forms and the production of AAV particles.Altogether, these results indicate that in the presence of HSV-1, AAV may replicate by using a basal set of cellular DNA replication enzymes but also relies extensively on HSV-1-derived proteins for its replication, including UL12, a newly discovered helper factor. These results suggest that AAV may be able to differentially adapt its replication strategy to the nuclear environment induced by the helper virus.     

The UL14 tegument protein of herpes simplex virus type 1 is required for efficient nuclear transport of the alpha transinducing factor VP16 and viral capsids

The protein encoded by the UL14 gene of herpes simplex virus type 1 (HSV-1) and HSV-2 is expressed late in infection and is a minor component of the virion tegument. An UL14-deficient HSV-1 mutant (UL14D) forms small plaques and exhibits an extended growth cycle at low multiplicities of infection (MOI) compared to wild-type virus. Although UL14 is likely to be involved in the process of viral maturation and egress, its precise role in viral replication is still enigmatic. In this study, we found that immediate-early viral mRNA expression was decreased in UL14D-infected cells. Transient coexpression of UL14 and VP16 in the absence of infection stimulated the nuclear accumulation of both proteins. We intended to visualize the fate of VP16 released from the infected virion and constructed UL14-null (14D-VP16G) and rescued (14R-VP16G) viruses that expressed a VP16-green fluorescent protein (GFP) fusion protein. Synchronous high-multiplicity infection of the viruses was performed at 4°C in the absence of de novo protein synthesis. We found that the presence of UL14 in the virion had an enhancing effect on the nuclear accumulation of VP16-GFP. The lack of UL14 did not significantly alter virus internalization but affected incoming capsid transport to the nuclear pore. These observations suggested that UL14 (i) enhanced VP16 nuclear localization at the immediately early phase, thus indirectly regulating the expression of immediate-early genes, and (ii) was associated with efficient nuclear targeting of capsids. The tegument protein UL14 could be part of the machinery that regulates HSV-1 replication.     

US3 of herpes simplex virus type 1 encodes a promiscuous protein kinase that phosphorylates and alters localization of lamin A/C in infected cells

The herpes simplex virus type 1 (HSV-1) US3 gene encodes a serine/threonine kinase that, when inactivated, causes capsids to aggregate aberrantly between the inner and outer nuclear membranes (INM and ONM, respectively) within evaginations/extensions of the perinuclear space. In both Hep2 cells and an engineered cell line derived from Hep2 cells expressing lamin A/C fused to enhanced green fluorescent protein (eGFP-lamin A/C), lamin A/C localized mostly in a reticular pattern with small regions of the INM devoid of eGFP-lamin A/C when they were either mock infected or infected with wild-type HSV-1(F). Cells infected with HSV-1(F) also contained some larger diffuse regions lacking lamin A/C. Proteins UL31 and UL34, markers of potential envelopment sites at the INM and perinuclear virions, localized within the regions devoid of lamin A/C and also in regions containing lamin A/C. Similar to previous observations with Vero cells (S. L. Bjerke and R. J. Roller, Virology 347:261-276, 2006), the proteins UL34 and UL31 localized exclusively in very discrete regions of the nuclear lamina lacking lamin A/C in the absence of US3 kinase activity. To determine how US3 alters lamin A/C distribution, US3 was purified and shown to phosphorylate lamin A/C at multiple sites in vitro, despite the presence of only one putative US3 kinase consensus site in the lamin A/C sequence. US3 kinase activity was also sufficient to invoke partial solubilization of lamin A/C from permeabilized Hep2 cell nuclei in an ATP-dependent manner. Two-dimensional electrophoretic analyses of lamin A/C revealed that lamin A/C is phosphorylated in HSV-infected cells, and the full spectrum of phosphorylation requires US3 kinase activity. These data suggest that US3 kinase activity regulates HSV-1 capsid nuclear egress at least in part by phosphorylation of lamin A/C.     

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.     

Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser²²). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser²² is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.     

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIα-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions −1 and −2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.     

Comparison of Effects of Inhibitors of Viral and Cellular Protein Kinases on Human Cytomegalovirus Disruption of Nuclear Lamina and Nuclear Egress

Human cytomegalovirus (HCMV) kinase UL97 is required for efficient nuclear lamina disruption during nuclear egress. However, cellular protein kinase C (PKC) has been implicated in this process in other systems. Comparing the effects of UL97 and cellular kinase inhibitors on HCMV nuclear egress confirms a role for UL97 in lamina disruption and nuclear egress. A pan-PKC inhibitor did not affect lamina disruption but did reduce the number of cytoplasmic capsids more than the number of nuclear capsids.     

The Differential Mobilization of Histones H3.1 and H3.3 by Herpes Simplex Virus 1 Relates Histone Dynamics to the Assembly of Viral Chromatin

During lytic infections, HSV-1 genomes are assembled into unstable nucleosomes. The histones required for HSV-1 chromatin assembly, however, are in the cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, and proposed that the mobilized histones are available for assembly into viral chromatin. However, the actual relevance of histone mobilization remained unknown. We now show that canonical H3.1 and variant H3.3 are also mobilized during HSV-1 infection. Mobilization required no HSV-1 protein expression, although immediate early or early proteins enhanced it. We used the previously known differential association of H3.3 and H3.1 with HSV-1 DNA to test the relevance of histone mobilization. H3.3 binds to HSV-1 genomes first, whereas H3.1 only binds after HSV-1 DNA replication initiates. Consistently, H3.3 and H3.1 were differentially mobilized. H3.1 mobilization decreased with HSV-1 DNA replication, whereas H3.3 mobilization was largely unaffected by it. These results support a model in which previously mobilized H3.1 is immobilized by assembly into viral chromatin during HSV-1 DNA replication, whereas H3.3 is mobilized and assembled into HSV-1 chromatin throughout infection. The differential mobilizations of H3.3 and H3.1 are consistent with their differential assembly into viral chromatin. These data therefore relate nuclear histone dynamics to the composition of viral chromatin and provide the first evidence that histone mobilization relates to viral chromatin assembly.     

The herpes simplex virus 1 protein kinase encoded by the US3 gene mediates posttranslational modification of the phosphoprotein encoded by the UL34 gene.

Earlier studies have shown that a herpes simplex virus 1 (HSV-1) open reading frame, US3, encodes a novel protein kinase and have characterized the cognate amino acid sequence which is phosphorylated by this enzyme. This report identifies an apparently essential viral phosphoprotein whose posttranslational processing involves the viral protein kinase. Analyses of viral proteins phosphorylated in the course of productive infection revealed a phosphoprotein whose mobility was viral protein kinase and serotype dependent. Thus, the corresponding HSV-1 and HSV-2 phosphoproteins differ in their electrophoretic mobilities, and the phosphoprotein specified by the HSV-1 mutant deleted in US3 (R7041) differs from that of the corresponding HSV-1 and HSV-2 proteins. Analyses of HSV-1 x HSV-2 recombinants mapped the phosphoprotein between 0.42 and 0.47 map units on the prototype HSV-1 DNA map. Within this region, the UL34 open reading frame was predicted to encode a protein of appropriate molecular weight which would also contain the consensus target site for phosphorylation by the viral protein kinase as previously defined with synthetic peptides. Replacement of the native UL34 gene with a UL34 gene tagged with a 17-amino-acid epitope from the alpha 4 protein identified this gene as encoding the phosphoprotein. Finally, mutagenesis of the predicted phosphorylation site on UL34 in the viral genome, and specifically the substitution of threonine or serine with alanine in the product of the UL34 gene, yielded phosphoproteins whose electrophoretic mobilities could not be differentiated from that of the US3- mutant. We conclude that the posttranslational processing of the UL34 gene product to its wild-type phenotype requires the participation of the viral protein kinase. While the viral protein kinase is not essential for viral replication in cells in culture, the UL34 gene product itself may not be dispensable.