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.     

UL31 of herpes simplex virus 1 is necessary for optimal NF-kappaB activation and expression of viral gene products

Previous results suggested that the U(L)31 gene of herpes simplex virus 1 (HSV-1) is required for envelopment of nucleocapsids at the inner nuclear membrane and optimal viral DNA synthesis and DNA packaging. In the current study, viral gene expression and NF-κB and c-Jun N-terminal kinase (JNK) activation of a herpes simplex virus mutant lacking the U(L)31 gene, designated ΔU(L)31, and its genetic repair construct, designated ΔU(L)31-R, were studied in various cell lines. In Hep2 and Vero cells infected with ΔU(L)31, expression of the immediate-early protein ICP4, early protein ICP8, and late protein glycoprotein C (gC) were delayed significantly. In Hep2 cells, expression of these proteins failed to reach levels seen in cells infected with ΔU(L)31-R or wild-type HSV-1(F) even after 18 h. The defect in protein accumulation correlated with poor or no activation of NF-κB and JNK upon infection with ΔU(L)31 compared to wild-type virus infection. The protein expression defects of the U(L)31 deletion mutant were not explainable by a failure to enter nonpermissive cells and were not complemented in an ICP27-expressing cell line. These data suggest that pU(L)31 facilitates initiation of infection and/or accelerates the onset of viral gene expression in a manner that correlates with NF-κB activation and is independent of the transactivator ICP27. The effects on very early events in expression are surprising in light of the fact that U(L)31 is designated a late gene and pU(L)31 is not a virion component. We show herein that while most pUL31 is expressed late in infection, low levels of pU(L)31 are detectable as early as 2 h postinfection, consistent with an early role in HSV-1 infection.     

Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.     

Conformational changes in the nuclear lamina induced by herpes simplex virus type 1 require genes U(L)31 and U(L)34

The herpes simplex virus type 1 (HSV-1) U(L)31 and U(L)34 proteins are dependent on each other for proper targeting to the nuclear membrane and are required for efficient envelopment of nucleocapsids at the inner nuclear membrane. In this work, we show that whereas the solubility of lamins A and C (lamin A/C) was not markedly increased, HSV induced conformational changes in the nuclear lamina of infected cells, as viewed after staining with three different lamin A/C-specific antibodies. In one case, reactivity with a monoclonal antibody that recognizes an epitope in the lamin tail domain was greatly reduced in HSV-infected cells. This apparent HSV-induced epitope masking required both U(L)31 and U(L)34, but these proteins were not sufficient to mask the epitope in uninfected cells, indicating that other HSV proteins are also required. In the second case, staining with a rabbit polyclonal antibody that primarily recognizes epitopes in the lamin A/C rod domain revealed that U(L)34 is required for HSV-induced decreased availability of epitopes for reaction with the antibody, whereas U(L)31 protein was dispensable for this effect. Still another polyclonal antibody indicated virtually no difference in lamin A/C staining in infected versus uninfected cells, indicating that the HSV-induced changes are more conformational than the result of lamin depletion at the nuclear rim. Further evidence supporting an interaction between the nuclear lamina and the U(L)31/U(L)34 protein complex includes the observations that (i) overexpression of the U(L)31 protein in uninfected cells was sufficient to relocalize lamin A/C from the nuclear rim into nucleoplasmic aggregates, (ii) overexpression of U(L)34 was sufficient to relocalize some lamin A/C into the cytoplasm, and (iii) both U(L)31 and U(L)34 could directly bind lamin A/C in vitro. These studies suggest that the U(L)31 and U(L)34 proteins modify the conformation of the nuclear lamina in infected cells, possibly by direct interaction with lamin A/C, and that other proteins are also likely involved. Given that the nuclear lamina potentially excludes nucleocapsids from envelopment sites at the inner nuclear membrane, the lamina alteration may reflect a role of the U(L)31/U(L)34 protein complex in perturbing the lamina to promote nucleocapsid egress from the nucleus. Alternatively, the data are compatible with a role of the lamina in targeting the U(L)31/U(L)34 protein complex to the nuclear membrane.     

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.     

U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U(L)31 protein fused to glutathione-S-transferase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demonstrated to specifically recognize the U(L)31 and U(L)34 proteins of approximately 34,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U(L)34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U(L)31 null virus. When transiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U(S)3-encoded protein kinase, previously shown to phosphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 kinase is required for even distribution of U(L)31 and U(L)34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U(L)31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)31 and U(L)34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment 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.     

Domain within herpes simplex virus 1 scaffold proteins required for interaction with portal protein in infected cells and incorporation of the portal vertex into capsids

The portal vertex of herpesvirus capsids serves as the conduit through which DNA is inserted during the assembly process. In herpes simplex virus (HSV), the portal is composed of 12 copies of the U(L)6 gene product, pU(L)6. Previous results identified a domain in the major capsid scaffold protein, ICP35, required for interaction with pU(L)6 and its incorporation into capsids formed in vitro (G. P. Singer et al., J. Virol. 74:6838-6848, 2005). In the current studies, pU(L)6 and scaffold proteins were found to coimmunoprecipitate from lysates of both HSV-infected cells and mammalian cells expressing scaffold proteins and pU(L)6. The coimmunoprecipitation of pU(L)6 and scaffold proteins was precluded upon deletion of codons 143 to 151 within U(L)26.5, encoding ICP35. While wild-type scaffold proteins colocalized with pU(L)6 when transiently coexpressed as viewed by indirect immunofluorescence, deletion of U(L)26.5 codons 143 to 151 precluded this colocalization. A recombinant herpes simplex virus, vJB11, was generated that lacked U(L)26.5 codons 143 to 151. A virus derived from this mutant but bearing a restored U(L)26.5 was also generated. vJB11 was unable to cleave or package viral DNA, whereas the restored virus packaged DNA normally. vJB11 produced ample numbers of B capsids in infected cells, but these lacked normal levels of pU(L)6. The deletion in U(L)26.5 also rendered pU(L)6 resistant to detergent extraction from vJB11-infected cells. These data indicate that, as was observed in vitro, amino acids 143 to 151 of ICP35 are critical for (i) interaction between scaffold proteins and pU(L)6 and (ii) incorporation of the HSV portal into capsids.     

The early region 1B 55-kilodalton oncoprotein of adenovirus relieves growth restrictions imposed on viral replication by the cell cycle.

The E1B 55-kDa oncoprotein of adenovirus enables the virus to overcome restrictions imposed on viral replication by the cell cycle. Approximately 20% of HeLa cells infected with an E1B 55-kDa mutant adenovirus produced virus when evaluated by electron microscopy or by assays for infectious centers. By contrast, all HeLa cells infected with a wild-type adenovirus produced virus. The yield of E1B mutant virus from randomly cycling HeLa cells correlated with the fraction of cells in S phase at the time of infection. In synchronously growing HeLa cells, approximately 75% of the cells infected during S phase with the E1B mutant virus produced virus, whereas only 10% of the cells infected during G1 produced virus. The yield of E1B mutant virus from HeLa cells infected during S phase was sevenfold greater than that of cells infected during G1 and threefold greater than that of cells infected during asynchronous growth. Cells infected during S phase with the E1B mutant virus exhibited severe cytopathic effects, whereas cells infected with the E1B mutant virus during G1 exhibited a mild cytopathic effect. Viral DNA synthesis appeared independent of the cell cycle because equivalent amounts of viral DNA were synthesized in cells infected with either wild-type or E1B mutant virus. The inability of the E1B mutant virus to replicate was not mediated by the status of p53. These results define a novel property of the large tumor antigen of adenovirus in relieving growth restrictions imposed on viral replication by the cell cycle.     

The herpes simplex virus 1 U(L)34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear membrane

To express the function encoded in its genome, the herpes simplex virus 1 capsid-tegument structure released by deenvelopment during entry into cells must be transported retrograde to the nuclear pore where viral DNA is released into the nucleus. This path is essential in the case of virus entering axons of dorsal root ganglia. The objective of the study was to identify the viral proteins that may be involved in the transport. We report the following findings. (i) The neuronal isoform of the intermediate chain (IC-1a) of the dynein complex pulled down, from lysates of [(35)S]methionine-labeled infected cells, two viral proteins identified as the products of U(L)34 and U(L)31 open reading frames, respectively. U(L)34 protein is a virion protein associated with cellular membranes and phosphorylated by the viral kinase U(S)3. U(L)31 protein is a largely insoluble, evenly dispersed nuclear phosphoprotein required for optimal processing and packaging of viral DNA into preformed capsids. Reciprocal pulldown experiments verified the interaction of IC-1a and U(L)34 protein. In similar experiments, U(L)34 protein was found to interact with U(L)31 protein and the major capsid protein ICP5. (ii) To determine whether U(L)34 protein is transported to the nuclear membrane, a requirement if it is involved in transport, the U(L)34 protein was inserted into a baculovirus vector under the cytomegalovirus major early promoter. Cells infected with the recombinant baculovirus expressed U(L)34 protein in a dose-dependent manner, and the U(L)34 protein localized primarily in the nuclear membrane. An unexpected finding was that U(L)34-expressing cells showed a dissociation of the inner and outer nuclear membranes reminiscent of the morphologic changes seen in cells productively infected with herpes simplex virus 1. U(L)34, like many other viral proteins, may have multiple functions expressed both early and late in infection.     

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.     

Herpes simplex virus type 1 alkaline nuclease is required for efficient processing of viral DNA replication intermediates.

Mutations in the alkaline nuclease gene of herpes simplex type 1 (HSV-1) (nuc mutations) induce almost wild-type levels of viral DNA; however, mutant viral yields are 0.1 to 1% of wild-type yields (L. Shao, L. Rapp, and S. Weller, Virology 195:146-162, 1993; R. Martinez, L. Shao, J.C. Bronstein, P.C. Weber, and S. Weller, Virology 215:152-164, 1996). nuc mutants are defective in one or more stages of genome maturation and appear to package DNA into aberrant or defective capsids which fail to egress from the nucleus of infected cells. In this study, we used pulsed-field gel electrophoresis to test the hypothesis that the defects in nuc mutants are due to the failure of the newly replicated viral DNA to be processed properly during DNA replication and/or recombination. Replicative intermediates of HSV-1 DNA from both wild-type- and mutant-infected cells remain in the wells of pulsed-field gels, while free linear monomers are readily resolved. Digestion of this well DNA with restriction enzymes that cleave once in the viral genome releases discrete monomer DNA from wild-type virus-infected cells but not from nuc mutant-infected cells. We conclude that both wild-type and mutant DNAs exist in a complex, nonlinear form (possibly branched) during replication. The fact that discrete monomer-length DNA cannot be released from nuc DNA by a single-cutting enzyme suggests that this DNA is more branched than DNA which accumulates in cells infected with wild-type virus. The well DNA from cells infected with wild-type and nuc mutants contains XbaI fragments which result from genomic inversions, indicating that alkaline nuclease is not required for mediating recombination events within HSV DNA. Furthermore, nuc mutants are able to carry out DNA replication-mediated homologous recombination events between inverted repeats on plasmids as evaluated by using a quantitative transient recombination assay. Well DNA from both wild-type- and mutant-infected cells contains free U(L) termini but not free U(S) termini. Various models to explain the structure of replicating DNA are considered.     

Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.     

The putative terminase subunit of herpes simplex virus 1 encoded by UL28 is necessary and sufficient to mediate interaction between pUL15 and pUL33

Viral terminases play essential roles as components of molecular motors that package viral DNA into capsids. Previous results indicated that the putative terminase subunits of herpes simplex virus 1 (HSV-1) encoded by U(L)15 and U(L)28 (designated pU(L)15 and pU(L)28, respectively) coimmunoprecipitate with the U(L)33 protein from lysates of infected cells. All three proteins are among six required for HSV-1 DNA packaging but dispensable for assembly of immature capsids. The current results show that in both infected- and uninfected-cell lysates, pU(L)28 coimmunoprecipitates with either pU(L)33 or pU(L)15, whereas pU(L)15 and pU(L)33 do not coimmunoprecipitate unless pU(L)28 is present. The U(L)28 protein was sufficient to stabilize pU(L)33 from proteasomal degradation in an engineered cell line and was necessary to stabilize pU(L)33 in infected cells, whereas pU(L)15 had no such effects. The presence of pU(L)33 was dispensable for the pU(L)15/pU(L)28 interaction in lysates of both infected and uninfected cells but augmented the tendency for pU(L)15 and pU(L)28 to coimmunoprecipitate. These data suggest that pU(L)28 and pU(L)33 interact directly and that pU(L)15 interacts directly with pU(L)28 but only indirectly with pU(L)33. It is logical to propose that the indirect interaction of pU(L)15 and pU(L)33 is mediated through the interaction of both proteins with pU(L)28. The data also suggest that one function of pU(L)33 is to optimize the pU(L)15/pU(L)28 interaction.     

The interaction of herpes simplex virus 1 regulatory protein ICP22 with the cdc25C phosphatase is enabled in vitro by viral protein kinases US3 and UL13

Earlier studies have shown that ICP22 and the U(L)13 protein kinase but not the U(S)3 kinase are required for optimal expression of a subset of late (gamma(2)) genes exemplified by U(L)38, U(L)41, and U(S)11. In primate cells, ICP22 mediates the disappearance of inactive isoforms of cdc2 and degradation of cyclins A and B1. Active cdc2 acquires a new partner, the viral DNA synthesis processivity factor U(L)42. The cdc2-U(L)42 complex recruits and phosphorylates topoisomerase IIalpha for efficient expression of the gamma(2) genes listed above. In uninfected cells, the cdc25C phosphatase activates cdc2 by removing two inhibitory phosphates. The accompanying report shows that in the absence of cdc25C, the rate of degradation of cyclin B1 is similar to that occurring in infected wild-type mouse embryo fibroblast cells but the levels of cdc2 increase, and the accumulation of a subset of late proteins and virus yields are reduced. This report links ICP22 with cdc25C. We show that in infected cells, ICP22 and U(S)3 protein kinase mediate the phosphorylation of cdc25C at its C-terminal domain. In in vitro assays with purified components, both U(L)13 and U(S)3 viral kinases phosphorylate cdc25C and ICP22. cdc25C also interacts with cdc2. However, in infected cells, the ability of cdc25C to activate cdc2 by dephosphorylation of the inactive cdc2 protein is reduced. Coupled with the phosphorylation of cdc25C by the U(S)3 kinase, the results raise the possibility that herpes simplex virus 1 diverts cdc25C to perform functions other than those performed in uninfected cells.     

Herpes simplex virus type 1 U(L)34 gene product is required for viral envelopment

The herpes simplex virus type 1 U(L)34 gene encodes a protein that is conserved in all human herpesviruses. The association of the U(L)34 protein with membranes in the infected cell and its expression as a gamma-1 gene suggest a role in maturation or egress of the virus particle from the cell. To determine the function of this gene product, we have constructed a recombinant virus that fails to express the U(L)34 protein. This recombinant virus, in which the U(L)34 protein coding sequence has been replaced by green fluorescent protein, forms minute plaques and replicates in single-step growth experiments to titers 3 to 5 log orders of magnitude lower than wild-type or repair viruses. On Vero cells, the deletion virus synthesizes proteins of all kinetic classes in normal amounts. Electron microscopic and biochemical analyses show that morphogenesis of the deletion virus proceeds normally to the point of formation of DNA-containing nuclear capsids, but electron micrographs show no enveloped virus particles in the cytoplasm or at the surface of infected cells, suggesting that the U(L)34 protein is essential for efficient envelopment of capsids.     

Lamins A and C but not lamin B1 regulate nuclear mechanics

Mutations in the nuclear envelope proteins lamins A and C cause a broad variety of human diseases, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. Cells lacking lamins A and C have reduced nuclear stiffness and increased nuclear fragility, leading to increased cell death under mechanical strain and suggesting a potential mechanism for disease. Here, we investigated the contribution of major lamin subtypes (lamins A, C, and B1) to nuclear mechanics by analyzing nuclear shape, nuclear dynamics over time, nuclear deformations under strain, and cell viability under prolonged mechanical stimulation in cells lacking both lamins A and C, cells lacking only lamin A (i.e. "lamin C-only" cells), cells lacking wild-type lamin B1, and wild-type cells. Lamin A/C-deficient cells exhibited increased numbers of misshapen nuclei and had severely reduced nuclear stiffness and decreased cell viability under strain. Lamin C-only cells had slightly abnormal nuclear shape and mildly reduced nuclear stiffness but no decrease in cell viability under strain. Interestingly, lamin B1-deficient cells exhibited normal nuclear mechanics despite having a significantly increased frequency of nuclear blebs. Our study indicates that lamins A and C are important contributors to the mechanical stiffness of nuclei, whereas lamin B1 contributes to nuclear integrity but not stiffness.     

Role of cellular phosphatase cdc25C in herpes simplex virus 1 replication

Earlier studies have shown that in herpes simplex virus 1-infected cells, ICP22 upregulates the accumulation of a subset of gamma(2) proteins exemplified by the products of the U(L)38, U(L)41, and U(S)11 genes. The ICP22-dependent process involves degradation of cyclins A and B1, the stabilization and activation of cdc2, physical interaction of activated cdc2 with the U(L)42 DNA synthesis processivity factor, and recruitment and phosphorylation of topoisomerase IIalpha by the cdc2/U(L)42 complex. Activation of cdc2, the first step in the process, is a key function of the mitotic phosphatase cdc25C. To define the role of cdc25C, we probed some features of the ICP22-dependent pathway of upregulation of gamma(2) genes in cdc25C(-/-) cells and in cdc25C(+/+) cells derived from sibling mice. We report that cyclin B1 turned over in cdc25C(+/+) or cdc25C(-/-) cells at the same rate, that cdc2 increased in amount, and that U(S)11 and U(L)38 proteins and infectious virus accumulated in smaller amounts than in wild-type infected cells. The reduction in U(L)38 protein accumulation and virus was greater in cdc25C(-/-) cells infected with virus lacking ICP22 than in cells infected with wild-type virus. We conclude that cdc25C phosphatase plays a role in viral replication and that this role extends beyond its function of activating cdc2 for initiation of the ICP22-dependent cascade for upregulation of gamma(2) gene expression.