A cellular protein related to heat-shock protein 90 accumulates during herpes simplex virus infection and is overexpressed in transformed cells

A monoclonal antibody produced from mice immunized with HSV-infected cell DNA binding proteins reacts with two cell-encoded polypeptides, p90 and p40, synthesized constitutively by many different cell types and expressed at high levels in transformed cells. In uninfected cells heat shock induces a cytoplasmic accumulation of p90 and in agreement with this we show that p90 is related to hsp90. During HSV-2 lytic productive infection, p90 accumulates to very high levels, such accumulation being detectable by 2 h p.i. In infected cells p90 is distributed throughout the cell, and in contrast to uninfected cells, is also located on the cell surface. Infection with HSV-1 strains causes an accumulation of p40, which has an intracellular location and is absent from the cell surface. These results show that HSV lytic infection results in an accumulation of at least two cell-encoded polypeptides, one of which is related to hsp90.     

T regulatory cells contribute to the attenuated primary CD8+ and CD4+ T cell responses to herpes simplex virus type 2 in neonatal mice

The first weeks of life are characterized by immune tolerance and increased susceptibility to intracellular pathogens. The neonatal adaptive response to HSV is attenuated compared with adult control models in humans and mice. T Regulatory cells (Tregs) control autoimmunity and excessive immune responses to infection. We therefore compared Treg responses in the draining lymph nodes (LN) of HSV-infected neonatal and adult C57BL/6 mice with the effect of Treg depletion/inactivation by anti-CD25 (PC61) treatment before infection on Ag-specific T cell effector responses at this site. There was a small, but significant increase in the frequency of CD4(+)Foxp3(+) Tregs at day 3 postinfection (p.i.) in the LN of neonatal and adult mice, compared with age-matched mock-infected controls. Depletion of Tregs before HSV infection significantly enhanced HSV-specific CD8(+) T cell cytotoxicity in vivo, cell number, activation, and granzyme B expression 4 days p.i. only in neonatal mice, and significantly enhanced CD8(+) and CD4(+) T cell IFN-gamma responses in both infected adults and neonates. Treg depletion also reduced the titer of infectious virus in the draining LN and nervous system of infected neonates on days 2 and 3 p.i. Treg suppression of the neonatal CTL response p.i. with HSV was associated with increased expression of TGF-beta in the draining LN at day 4 p.i. compared with uninfected neonates, but IL-10 was increased in infected adults alone. These experiments support the notion that the newborn primary T cell effector responses to HSV are suppressed by Tregs.     

A major heat-shock protein defined by a monoclonal antibody.

A monoclonal antibody reacts with a polypeptide of 68 000 mol. wt. (p68) that accumulates to high levels during heat shock. The intracellular distribution of this antigen in normal and heat-shocked cells has been studied. It is a major component of non-stressed cells, where it is located predominantly in the cytoplasm, but also occurs in the nucleus. The nuclear accumulation is growth regulated, in that exponentially growing cells have strong nuclear immunofluorescence and confluent cells little. It is concentrated at the leading edge of motile fibroblasts and co-distributes with actin-containing microfilaments. Heat shock causes cytoplasmic and nuclear accumulation and there is new deposition in the periphery of cells. In normal cells the antigen in the nucleus is located in the nuclear lamina and matrix which increases during heat shock. The distribution of this molecule and the structures with which it interacts suggests that it is important in mediating the effects of heat shock.     

Cellular proteins expressed in herpes simplex virus transformed cells also accumulate on herpes simplex virus infection.

The cell proteins expressed in rat embryo cells transformed by herpes simplex virus (HSV) have been analysed by immunoprecipitation assays to determine those polypeptides which can be identified by immunoprecipitation with the sera of tumour-bearing animals and also with antisera to herpes simplex infected cells. Cell polypeptides commonly recognised by both these sera have been further characterised using a monoclonal antibody directed against a cellular polypeptide which accumulates on HSV-2 lytic infection. This monoclonal antibody recognises in HSV-transformed cells polypeptides of mol. wts. 90 000, 40 000 and 32 000. Further studies show that the accumulation of these polypeptides in HSV-transformed cells is not HSV specific but is a common feature of transformation or of cells which have been immortalised. We suggest that cellular polypeptides accumulating as a result of HSV infection may be of importance in the initiation of transformation by HSV, i.e., at the level of immortalisation of cells.     

Identification of a Golgi-associated protein that undergoes mitosis dependent phosphorylation and relocation

By means of a monoclonal antibody (BH3), we have identified a 57-kD protein (p57) that in interphase is restricted largely to the perinuclear region of the cell. Double label immunofluorescence microscopy suggests localization of p57 to the Golgi complex and associated membranous structures. Protease protection experiments and chemical extractability indicate that p57 is a peripheral membrane protein exposed to the cytoplasm. p57 displays unique behavior during mitosis. At the end of G2 or in early prophase, p57 leaves the perinuclear region and accumulates very rapidly within the nucleus, at a time when the nuclear envelope is still intact and before nuclear lamina disassembly. This relocation of p57 coincides with its hyperphosphorylation on serine and threonine residues. After nuclear envelope breakdown p57 becomes uniformly distributed throughout the mitotic cytoplasm until in late telophase when it returns to its perinuclear location and is once again excluded from the nucleus. The behavior of p57 during mitosis suggests that it may play a role in the cellular reorganization evident during mitotic prophase.     

Herpes simplex virus type-1-induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion

Adaptive cellular immunity is required to clear HSV-1 infection in the periphery. Myeloid dendritic cells (DCs) are the first professional Ag-presenting cell to encounter the virus after primary and secondary infection and thus the consequences of their infection are important in understanding the pathogenesis of the disease and the response to the virus. Following HSV-1 infection, both uninfected and infected human DCs acquire a more mature phenotype. In this study, we demonstrate that type I IFN secreted from myeloid DC mediates bystander activation of the uninfected DCs. Furthermore, we confirm that this IFN primes DCs for elevated IL-12 p40 and p70 secretion. However, secretion of IFN is not responsible for the acquisition of a mature phenotype by HSV-1-infected DC. Rather, virus binding to a receptor on the cell surface induces DC maturation directly, through activation of the NF-kappaB and p38 MAPK pathways. The binding of HSV glycoprotein D is critical to the acquisition of a mature phenotype and type I IFN secretion. The data therefore demonstrate that DCs can respond to HSV exposure directly through recognition of viral envelope structures. In the context of natural HSV infection, the coupling of viral entry to the activation of DC signaling pathways is likely to be counterbalanced by viral disruption of DC maturation. However, the parallel release of type I IFN may result in paracrine activation so that the DCs are nonetheless able to mount an adaptive immune response.     

Explant-induced reactivation of herpes simplex virus occurs in neurons expressing nuclear cdk2 and cdk4

Herpes simplex virus (HSV) establishes productive (lytic) infections in nonneuronal cells and nonproductive (latent) infections in neurons. It has been proposed that HSV establishes latency because quiescent neurons lack cellular factors required for productive infection. It has been further proposed that these putative factors are induced following neuronal stress, as a requirement for HSV reactivation. To date, the identity of these putative cellular factors remains unknown. We have demonstrated that cyclin-dependent kinase (cdk) 1, 2, or 7 is required for HSV replication in nonneuronal cells. Interestingly, cdks 1 and 2 are not expressed in quiescent neurons but can be induced in stressed neurons. Thus, cdks may be among the cellular proteins required for HSV reactivation whose neuronal expression is differentially regulated during stress. Herein, we determined that neuronal expression of nuclear cdk2, cdk4, and cyclins E and D2 (which activate cdks 2 and 4, respectively) was induced following explant cultivation, a stressful stimulus that induces HSV reactivation. In contrast, neuronal expression of cdk7 and cytoplasmic cdk4 decreased during explant cultivation, whereas cdk3 was detected in the same small percentage of neurons before and after explant cultivation and cdks 1, 5, and 6 were not detected in neuronal cell bodies. HSV-1 reactivated specifically in neurons expressing nuclear cdk2 and cdk4, and an inhibitor specific for cdk2 inhibited HSV-1 reactivation. We conclude that neuronal levels of cdk2 are among the factors that determine the outcome of HSV infections of neurons.     

Herpes simplex virus glycoprotein K is known to influence fusion of infected cells, yet is not on the cell surface.

Syncytial mutants of herpes simplex virus (HSV) cause extensive fusion of cultured cells, whereas wild-type HSV primarily causes cell rounding and aggregation. A large fraction of syncytial viruses contain mutations in the UL53 gene, which encodes glycoprotein K (gK). Previously, we demonstrated that wild-type and syncytial forms of gK are expressed at similar levels and possess identical electrophoretic mobilities. Using immunofluorescence, we show that gK is not transported to the surfaces of cells infected with either wild-type or syncytial HSV. Instead, gK accumulates in the perinuclear and nuclear membranes of cells. This finding is in contrast to the behavior of all other HSV glycoproteins described to date, which reach the cell surface. When gK was expressed in the absence of other HSV proteins, using a recombinant adenovirus vector, a similar perinuclear and nuclear pattern was observed. In addition, gK remained sensitive to endoglycosidase H, consistent with the hypothesis that gK does not reach the Golgi apparatus and is retained in the endoplasmic reticulum and nuclear envelope. Therefore, although gK mutations promote fusion between the surface membranes of HSV-infected cells, the glycoprotein does not reach the plasma membrane and, thus, must influence fusion indirectly.     

Nuclear accumulation of a heat-shock 70-like protein during herpes simplex virus replication

A monoclonal antibody defines an antigen, p68, related to hsp70, which is located in nuclei of uninfected exponential cells. Nuclear p68 is released by DNase but not RNase treatment suggesting an association with DNA. Lytic productive infection of confluent quiescent BHK 21 cells with herpes simplex virus type-2 causes p68 to accumulate in nuclei. The effect is specific for HSV-2, and does not occur in HSV-1 infected cells. Maximum nuclear accumulation of p68 requires virus DNA synthesis although a significant accumulation occurs in the absence of such synthesis. It is suggested that the nuclear accumulation of p68 is an aspect of a cellular stress response to lytic infection with HSV-2.Imperial Cancer Research Fund, Tumour Immunology Unit.     

Roles of tumor necrosis factor alpha (TNF-alpha) and the p55 TNF receptor in CD1d induction and coxsackievirus B3-induced myocarditis

Giving C57BL/6 mice 10(4) PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 10(5) or 10(6) PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 10(4) PFU were reduced. Tumor necrosis factor alpha (TNF-alpha) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-alpha(-/-) and p55 TNF receptor-negative (TNFR(-/-)) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR(-/-) mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR(-/-) animals showed 10-fold more inflammatory cells in the heart than p55 TNFR(-/-) mice, and the cell population was comprised of high concentrations of CD4(+) gamma interferon-positive and Vgamma4(+) cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR(-/-) mice upregulate CD1d, the molecule recognized by Vgamma4(+) cells, but infection of TNF(-/-) or p55 TNFR(-/-) endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-alpha, failed to elicit CD1d expression, but TNF-alpha treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-alpha treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-alpha signaling.     

Monoclonal antibody-aided characterization of cellular p220 in uninfected and poliovirus-infected HeLa cells: subcellular distribution and identification of conformers.

A monoclonal antibody directed against the Mr-220,000 subunit (p220) of the mRNA cap-binding complex has been prepared and used to analyze the sucrose gradient sedimentation and subcellular location of p220 and its poliovirus-induced cleavage products. The antibody reacted with p220 on immunoblots of cell lysates from uninfected cells, but only with several smaller polypeptides, the p220 cleavage products, in cell lysates from poliovirus-infected cells. The sedimentation of p220 antigens from uninfected or infected cells was analyzed by immunoblot and by enzyme-linked immunosorbent assay (ELISA) of sucrose gradient fractions. The results indicate that antibody reactivity was partially influenced by antigen conformation. Major forms of intact p220 and cleaved p220 were identified by immunoblot, and these had similar sedimentation properties. ELISA analysis of the same gradient fractions detected only uncleaved p220; p220 cleavage products were not recognized. Furthermore, the antibody recognized two forms of native uncleaved p220, one of which appeared to bind antibody with greater affinity. This result suggested the existence of conformational variants of p220. The differential reactivity of the antibody for cleaved versus uncleaved p220 served as a useful control during indirect immunofluorescence analysis to determine the subcellular distribution of p220 antigens. The distribution of p220 in uninfected cells was mainly cytoplasmic, but some nuclear antigens were also apparent. After poliovirus infection only the nuclear pattern remained. Disappearance of the cytoplasmic pattern confirmed the inability of the antibody to react with native p220 cleavage products. The cytoplasmic pattern also disappeared after human rhinovirus 14 infection, but not after mengovirus infection, results which correlated with the ability of human rhinovirus 14 and the inability of mengovirus to induce the cleavage of p220. The results demonstrate that p220 is not likely to be associated with the cytoskeleton and hint at the possibility of a partially nuclear location.     

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.     

Innate resistance to herpes simplex virus infection. Human lymphocyte and monocyte inhibition of viral replication

Peripheral blood monocytes and lymphocytes isolated from most humans are resistant to HSV infection in vitro. Viral replication is inhibited very early in the cycle, prior to the onset of alpha-protein synthesis; no viral protein or DNA synthesis is detectable even up to 1 week later. The enhanced expression of two 62-kDa and 57-kDa cellular proteins, however, is induced in the lymphocyte population within 3 to 5 h after infection. A 30-kDa protein is induced in the monocyte population immediately after infection. The induced expression of 62-kDa and 57-kDa lymphocyte proteins appears to be virus-mediated because: a) HSV and pseudorabies virus (although not vaccinia virus) induce the expression of 62-kDa and 57-kDa proteins, b) heat shock or exposure of lymphocytes to uninfected cell extracts does not induce expression of either protein, c) 62-kDa protein is not induced in lymphocytes stimulated with a mitogenic concentration of PHA. UV-inactivated HSV induces expression of 62-kDa and 57-kDa proteins in a manner similar to that observed with untreated virus. In contrast, expression of 30-kDa monocyte protein is induced nonspecifically by either uninfected cell extracts or cell extracts containing virus. Sixty-two-kilodalton and 57-kDa protein induction appears to be a marker for human lymphocytes that express profound intracellular resistance to infection with HSV. Induced expression of these proteins occurs only in lymphocytes that inhibit viral replication very early in the growth cycle, prior to the onset of alpha-protein synthesis. Expression of 62-kDa and 57-kDa proteins is not induced in lymphocytes that are permissive or partially permissive to infection with HSV.     

Cells expressing herpes simplex virus glycoprotein gC but not gB, gD, or gE are recognized by murine virus-specific cytotoxic T lymphocytes

To determine which viral molecule(s) is recognized by herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL), target cells were constructed which express individual HSV glycoproteins. A mouse L cell line, Z4/6, which constitutively expressed high levels of HSV type 2 (HSV-2) gD (gD-2) was isolated and characterized previously (D. C. Johnson and J. R. Smiley, J. Virol. 54:682-689, 1985). Despite the expression of gD on the surface of Z4/6 cells, these cells were not killed by anti-HSV-2 CTL generated following intravaginal infection of syngeneic mice. In contrast, parental Z4 or Z4/6 cells infected with HSV-2 were lysed. Furthermore, unlabeled Z4/6 cells were unable to block the lysis of HSV-2-infected labeled target cells. Cells which express HSV-1 gB (gB-1) were isolated by transfecting L cells with the recombinant plasmid pSV2gBneo, which contains the HSV-1 gB structural sequences and the neomycin resistance gene coupled to the simian virus 40 early promoter and selecting G418-resistant cell lines. One such cell line, Lta/gB15, expressed gB which was detected by immunoprecipitation and at the cell surface by immunofluorescence. Additionally, cells expressing HSV-1 gC (gC-1) or gE (gE-1) were isolated by transfecting Z4 cells, which are L cells expressing ICP4 and ICP47, with either the recombinant plasmid pGE15neo, which contains the gE structural sequences and the neomycin resistance gene, or pDC17, which contains the gC structural gene coupled to the gD-1 promoter. A number of G418-resistant cell lines were isolated which expressed gC-1 or gE-1 at the cell surface. Anti-HSV-1 CTL generated following footpad infection of syngeneic mice were unable to lyse target cells expressing gB-1 or gE-1. In contrast, target cells expressing very low levels of gC-1 were killed as well as HSV-1-infected target cells. Furthermore, infection of gC-1-transformed target cells with wild-type HSV-1 or a strain of HSV-1 that does not express gC did not result in a marked increase in susceptibility to lysis. These results suggest that murine class I major histocompatibility complex-restricted anti-HSV CTL recognize gC-1 but do not recognize gB, gD, or gE as these molecules are expressed in transfected syngeneic target cells. The results are discussed in terms of recent evidence concerning the specificity of antiviral CTL.     

Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of 51Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a+ Leu-4- granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of 51Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.     

Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein.

We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein.     

Gene expression of herpes simplex virus. I. Analysis of cytoplasmic RNAs in infected xeroderma pigmentosum cells.

RNAs which are synthesized and accumulate in the cytoplasm of uninfected and herpes simplex virus type 1 (HSV-1)-infected xeroderma pigmentosum (XP) cells in the presence of cycloheximide (early RNAs) or absence of drugs (late RNAs) were analyzed by electrophoresis through denaturing polyacrylamide gradient slab gels. HSV RNAs were selected by hybridization ot HSV DNA covalently bound to cellulose. No HSV-specific low-molecular-weight (4S to 10S) RNAs were detected. However, several changes were observed in the electrophoretic pattern of the host low-molecular-weight RNAs during HSV infection. Five HSV RNAs ranging in size from 16S to 28S accumulated in the cytoplasm of infected XP cells in the presence of cycloheximide. These are of the size range predicted to encode the major early viral polypeptides. The cytoplasmic and polyadenylated early RNAs from HSV-infected XP cells were translated in vitro to produce proteins whose electrophoretic pattern resembled that of the early viral proteins synthesized in vivo.     

Fate of the inner nuclear membrane protein lamin B receptor and nuclear lamins in herpes simplex virus type 1 infection

During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B(2) tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B(1), and B(2). By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane.     

Negative regulation of mitotic promoting factor by the checkpoint kinase chk1 in simian virus 40 lytic infection

Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G(1)/S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G(1)/S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G(1)/S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G(2). Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.     

The cellular localization pattern of Varicella-Zoster virus ORF29p is influenced by proteasome-mediated degradation

Varicella-zoster virus (VZV) open reading frame 29 (ORF29) encodes a single-stranded DNA binding protein. During lytic infection, ORF29p is localized primarily to infected-cell nuclei, whereas during latency it appears in the cytoplasm of infected neurons. Following reactivation, ORF29p accumulates in the nucleus. In this report, we analyze the cellular localization patterns of ORF29p during VZV infection and during autonomous expression. Our results demonstrate that ORF29p is excluded from the nucleus in a cell-type-specific manner and that its cellular localization pattern may be altered by subsequent expression of VZV ORF61p or herpes simplex virus type 1 ICP0. In these cases, ORF61p and ICP0 induce nuclear accumulation of ORF29p in cell lines where it normally remains cytoplasmic. One cellular system utilized by ICP0 to influence protein abundance is the proteasome degradation pathway. Inhibition of the 26S proteasome, but not heat shock treatment, resulted in accumulation of ORF29p in the nucleus, similar to the effect of ICP0 expression. Immunofluorescence microscopy and pulse-chase experiments reveal that stabilization of ORF29p correlates with its nuclear accumulation and is dependent on a functional nuclear localization signal. ORF29p nuclear translocation in cultured enteric neurons and cells derived from an astrocytoma is reversible, as the protein's distribution and stability revert to the previous states when the proteasomal activity is restored. Thus, stabilization of ORF29p leads to its nuclear accumulation. Although proteasome inhibition induces ORF29p nuclear accumulation, this is not sufficient to reactivate latent VZV or target the immediate-early protein ORF62p to the nucleus in cultured guinea pig enteric neurons.