Levels Of Viral DNA Synthesis Research Articles

Nucleolar Phosphoprotein NPM1 Interacts With Porcine Circovirus Type 3 Cap Protein and Facilitates Viral Replication

Porcine circovirus type 3 (PCV3) is a recently discovered virus with potentially significant implications on the global swine industry. PCV3 replication involves the entry of the viral capsid (Cap) protein with nucleolar localization signals into the nucleus. Using liquid chromatography-mass spectrometry analysis, nucleolar phosphoprotein NPM1 was identified as one of the cellular proteins bound to PCV3 Cap. Co-immunoprecipitation demonstrated that PCV3 Cap interacts directly with NPM1, where the region binding with NPM1 is mapped to amino acid residues 1–38 of Cap. Upon co-transfection, the expression of Cap protein promoted the redistribution of NPM1, which translocated from the nucleus to the cytoplasm and colocalized with Cap in cultured PK15 cells. NPM1 expression was upregulated and translocated from the nucleus to the cytoplasm in PCV3-infected cells, upon siRNA-mediated depletion, or upon treatment with NPM1 inhibitor in PK15 cells with impaired PCV3 replication, as evidenced by decreased levels of viral DNA synthesis and protein expression. By contrast, the replication of PCV3 was enhanced in stably NPM1-expressing cells via a lentivirus-delivered system. Taken together, these findings indicate that NPM1 interacts with PCV3 Cap and plays a crucial role in PCV3 replication.

Cellular v-ATPase is required for virion assembly compartment formation in human cytomegalovirus infection.

Successful generation of virions from infected cells is a complex process requiring orchestrated regulation of host and viral genes. Cells infected with human cytomegalovirus (HCMV) undergo a dramatic reorganization of membrane organelles resulting in the formation of the virion assembly compartment, a process that is not fully understood. Here we show that acidification of vacuoles by the cellular v-ATPase is a crucial step in the formation of the virion assembly compartment and disruption of acidification results in mis-localization of virion components and a profound reduction in infectious virus levels. In addition, knockdown of ATP6V0C blocks the increase in nuclear size, normally associated with HCMV infection. Inhibition of the v-ATPase does not affect intracellular levels of viral DNA synthesis or gene expression, consistent with a defect in assembly and egress. These studies identify a novel host factor involved in virion production and a potential target for antiviral therapy.

The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

We report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb' region of the viral genome. A UL97-null (Δ97) mutant of strain TB40/E, which encodes a full-length ULb' region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a Δ97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb' region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(β-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb' regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ≈ 100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb' regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb' region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb' region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis.

A Hsp40 Chaperone Protein Interacts with and Modulates the Cellular Distribution of the Primase Protein of Human Cytomegalovirus

Genomic DNA replication is a universal and essential process for all herpesvirus including human cytomegalovirus (HCMV). HCMV UL70 protein, which is believed to encode the primase activity of the viral DNA replication machinery and is highly conserved among herpesviruses, needs to be localized in the nucleus, the site of viral DNA synthesis. No host factors that facilitate the nuclear import of UL70 have been reported. In this study, we provided the first direct evidence that UL70 specifically interacts with a highly conserved and ubiquitously expressed member of the heat shock protein Hsp40/DNAJ family, DNAJB6, which is expressed as two isoforms, a and b, as a result of alternative splicing. The interaction of UL70 with a common region of DNAJB6a and b was identified by both a two hybrid screen in yeast and coimmunoprecipitation in human cells. In transfected cells, UL70 was primarily co-localized with DNAJB6a in the nuclei and with DNAJB6b in the cytoplasm, respectively. The nuclear import of UL70 was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Furthermore, the level of viral DNA synthesis and progeny production was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Thus, DNAJB6a and b appear to enhance the nuclear import and cytoplasmic accumulation of UL70, respectively. Our results also suggest that the relative expression levels of DNAJB6 isoforms may play a key role in regulating the cellular localization of UL70, leading to modulation of HCMV DNA synthesis and lytic infection.

Phosphorylation of p27Kip1 by Epstein-Barr Virus Protein Kinase Induces Its Degradation through SCFSkp2 Ubiquitin Ligase Actions during Viral Lytic Replication

Epstein-Barr virus (EBV) productive replication occurs in an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. The EBV protein kinase (PK), encoded by the viral BGLF4 gene, is a Ser/Thr protein kinase, which phosphorylates both viral and cellular proteins, modifying the cellular environment for efficient viral productive replication. We here provide evidence that the EBV PK phosphorylates the CDK inhibitor p27(Kip1), resulting in ubiquitination and degradation in a proteasome-dependent manner during EBV productive replication. Experiments with BGLF4 knockdown by small interfering RNA and BGLF4 knock-out viruses clarified that EBV PK is involved in p27(Kip1) degradation upon lytic replication. Transfection of the BGLF4 expression vector revealed that EBV PK alone could phosphorylate the Thr-187 residue of p27(Kip1) and that the ubiquitination and degradation of p27(Kip1) occurred in an SCF(Skp2) ubiquitin ligase-dependent manner. In vitro, EBV PK proved capable of phosphorylating p27(Kip1) at Thr-187. Unlike cyclin E-CDK2 activity, the EBV PK activity was not inhibited by p27(Kip1). Overall, EBV PK enhances p27(Kip1) degradation effectively upon EBV productive replication, contributing to establishment of an S-phase-like cellular environment with high CDK activity.

Productive replication of Malacosoma neustria nucleopolyhedrovirus (ManeNPV) in Md203 cell line

A plaque-purified genotypic variant, derived from a field isolate of the Malacosoma neustria nucleopolyhedrovirus (ManeNPV) from Turkey was characterized based on in vitro replication properties in a cell line, Md203 derived from Malacosoma disstria. The life cycle of ManeNPV was studied based on the cytopathic effects (CPEs), polyhedral inclusion body (PIB) formation, budded virus (BV) production, viral DNA replication and polyhedrin protein expression in ManeNPV-infected Md203 cells. Infection of Md203 cells with ManeNPV resulted in cytopathic effects within 24 and 36 h post infection and most cells detachted from the bottom of the culture plate on the fourth day. Typical NPV cytopathic effects (CPE) like granulated and rounded cells, nuclear hypertrophy and impairment in cell proliferation, and lost cell shape with the extendings were observed. Budded viruses were detected at 24 h p.i. However, significant increase in BV production was observed after 48 h p.i. PIB was firstly detected at 36 h p.i. When BV’s were used for reinfection of the Md203 cells, the result showed that these cells support production of viable virus progeny. We also observed a significant level of viral DNA synthesis by 24 h p.i. The results indicate that ManeNPV can be propagated at in vitro system for further studies and thus has a potential for development into an effective biotechnological and microbial insecticidal agent.

The Late Promoter of the Human Cytomegalovirus Viral DNA Polymerase Processivity Factor Has an Impact on Delayed Early and Late Viral Gene Products but Not on Viral DNA Synthesis

Transcription of the DNA polymerase processivity factor gene (UL44) of human cytomegalovirus initiates at three distinct start sites, which are differentially regulated during productive infection. Two of these start sites, the distal and proximal sites, are active at early times, and the middle start site is active at only late times after infection (F. Leach and E. S. Mocarski, J. Virol. 63:1783-1791, 1989). Compared to the wild type, UL44 gene expression was lower for recombinant viruses with the distal or the middle TATA element mutated. The transcripts initiating from the distal or middle start site facilitated late viral gene expression. The level of viral DNA synthesis was affected by mutation of the distal TATA element. In contrast, mutation of the middle TATA element did not affect the level of viral DNA synthesis, but it did affect significantly the level of late viral gene expression. Recombinant viruses with the distal or middle TATA element mutated grew more slowly than the wild type at both low and high multiplicities of infection. Reduced expression of the UL44 gene from the late middle viral promoter correlated with decreased late viral protein expression and decreased viral growth.

Herpes Simplex Virus DNA Synthesis Is Not a Decisive Regulatory Event in the Initiation of Lytic Viral Protein Expression in Neurons In Vivo during Primary Infection or Reactivation from Latency

The herpes simplex virus genome can enter a repressed transcriptional state (latency) in sensory neurons of the host nervous system. Although reduced permissiveness of the neuronal environment is widely accepted as a causal factor, the molecular pathway(s) directing and maintaining the viral genome in the latent state remains undefined. Over the past decade, the field has been strongly influenced by the observations of Kosz-Vnenchak et al., which have been interpreted to indicate that, in sensory neurons in vivo, a critical level of viral DNA synthesis within the neuron is required for sufficient viral immediate-early (IE) and early (E) gene expression (M. Kosz-Vnenchak, J. Jacobson, D. M. Coen, and D. M. Knipe, J. Virol. 67:5383-5393, 1993). The levels of IE and E genes are, in turn, thought to regulate the decision to enter the lytic cycle or latency. We have reexamined this issue using new strategies for in situ detection and quantification of viral gene expression in whole tissues. Our results using thymidine kinase-null and rescued mutants as well as wild-type strains in conjunction with viral DNA synthesis blockers demonstrate that (i) despite inhibition of viral DNA replication, many neurons express lytic viral proteins, including IE proteins, during acute infection in the ganglion; (ii) at early times postinoculation, the number of neurons expressing viral proteins in the ganglion is not reduced by inhibition of viral DNA replication; and (iii) following a reactivation stimulus, the numbers of neurons and apparent levels of lytic viral proteins, including IE proteins, are not reduced by inhibition of viral DNA replication. We conclude that viral DNA replication in the neuron per se does not regulate IE gene expression or entry into the lytic cycle.

Somatic cell mutants resistant to retrovirus replication: intracellular blocks during the early stages of infection.

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

The herpes simplex virus type 1 cleavage/packaging protein, UL32, is involved in efficient localization of capsids to replication compartments.

Six genes, including UL32, have been implicated in the cleavage and packaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isolated temperature-sensitive herpes simplex virus type 1 mutant, tsN20 (P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virology 52:57-71, 1973). A polyclonal antibody which recognizes UL32 was previously used by Chang et al. (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938-3946, 1996) to demonstrate that UL32 accumulates predominantly in the cytoplasm of infected cells. In this report, a functional epitope-tagged version of UL32 showed that while UL32 is predominantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICP8, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific for the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results suggest that cleavage and packaging occur in replication compartments at least at 6 and 8 h postinfection. Cells infected with the UL32 mutant exhibit a hexon staining pattern which is more diffusely distributed throughout the nucleus and which is not restricted to replication compartments. We propose that UL32 may play a role in "bringing" preassembled capsids to the sites of DNA packaging and that the failure to localize to replication compartments may explain the cleavage/packaging defect exhibited by this mutant. These results suggest that the UL32 protein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correct localization of capsids within infected cells.

The small non-structural protein NS2 of the autonomous parvovirus minute virus of mice is required for virus growth in murine cells.

Mutants of the autonomous parvovirus minute virus of mice (MVM) strains MVM(p) and MVM(i) that either fail to produce or produce a truncated NS2 protein, were deficient in the production of infectious virus and attained lower levels of viral DNA synthesis than wild-type virus following infection of a series of normal and transformed murine cell lines. Mutant virus growth and the levels of DNA replication were similar to those of wild-type virus in the rat, hamster and human lines tested. These results suggest that the requirement of NS2 for the growth of MVM is murine species-specific.

Deletion of the VP16 open reading frame of herpes simplex virus type 1.

VP16 (also called Vmw65 and alpha TIF) is a structural protein of herpes simplex virus type 1 (HSV-1) that trans-induces HSV-1 immediate-early gene transcription. This report describes an HSV-1 VP16 deletion mutant that was constructed and propagated in a cell line transformed with a VP16 expression vector. The VP16 deletion mutant replicated like wild-type HSV-1 during infection of the VP16-expressing cell line. Deletion mutant virions propagated in this cell line contained wild-type, cell-derived VP16 protein that was recruited during virion assembly and was functional for immediate-early gene trans-induction. The mutant failed to replicate during subsequent infection of cells that do not express VP16, as determined in plaque assays and single-step replication assays. The deletion mutant induced nearly normal levels of viral DNA synthesis and capsid production during these infections, but it induced slightly lower levels of viral DNA encapsidation and appeared by transmission electron microscopy to be defective in further steps of virion maturation. A genetic revertant of the deletion mutant that was restored for VP16-coding sequences exhibited fully wild-type replication properties in both VP16-expressing and nonexpressing cells. The absence of VP16 protein synthesis at late times of HSV-1 infection prevents the production of infectious progeny virus and correlates with a profound defect in HSV-1 particle assembly.

Cis-acting sequences that control the level of viral DNA synthesis in the polyomavirus late region

A deletion in the polyomavirus late region results in a drastic reduction of viral replication, as shown after transfection of viral DNA into 3T6 cells. This mutation is cis acting, since cotransfection with wild-type DNA did not restore the normal phenotype. Viral DNA synthesis returned to normal levels only after reintroduction of the authentic sequences in either orientation. The data presented here suggest that these sequences are involved in the binding of a factor(s) that controls the level of viral replication.

Effect of macrophage activation on resistance of mouse peritoneal macrophages to infection with herpes simplex virus types 1 and 2.

To define the effect of heterogeneity of murine peritoneal macrophages (M phi) on intrinsic resistance to herpes simplex virus (HSV) infection, several M phi populations were characterized for their response to infection with HSV type 1 (HSV-1) and HSV-2. Steady-state resident M phi (Res M phi) were compared in parallel with M phi activated with Corynebacterium parvum (now designated Propionibacterium acnes) (CP M phi) and thioglycollate-elicited inflammatory M phi (TG M phi). Res M phi were completely non-permissive for productive virus infection and showed no c.p.e. The intrinsic resistance of CP M phi to HSV infection was similar to that of Res M phi, in that the infection was non-productive for infectious virus, but CP M phi showed marked c.p.e. TG M phi showed semi-permissiveness, with virus yields at least 10-fold higher than those in Res M phi and CP M phi, and marked c.p.e. The three distinct intrinsic response patterns were maintained regardless of whether M phi were derived from CD-1 or B6C3F1 mice, or whether the infecting virus was HSV-1 or HSV-2. To define the level at which M phi restrict HSV replication, immunofluorescence assays for viral antigens and hybridization analyses for viral DNA were performed. All M phi populations showed immediate early and early virus polypeptides. Res M phi and CP M phi showed no viral DNA replication, but TG M phi showed moderate levels of viral DNA synthesis that paralleled the infectious virus titres produced. Investigation of the mechanism for the heterogeneous intrinsic antiviral response among the M phi revealed that interferon was not involved, because antiserum to mouse alpha/beta interferon did not alter the intrinsic resistance patterns. Induction of c.p.e. in M phi required live, replication-competent HSV. The involvement of tumour necrosis factor (TNF) in c.p.e. was found to be unlikely; no significant amounts of TNF were detected in the culture medium of the M phi, and inclusion of anti-TNF antibody did not inhibit c.p.e.

Temperature-sensitive herpes simplex virus type 1 mutants defective in the shutoff of cellular DNA synthesis and host polypeptide synthesis.

Two temperature-sensitive herpes simplex virus type 1 mutants, ts 1-8 and ts 199, belonging to different complementation groups, were isolated. Both mutants were defective in the shutoff of host DNA synthesis at 39.5 degrees C (nonpermissive temperature). ts 1-8 exhibited intermediate levels of viral DNA synthesis at 39.5 degrees C, while ts 199 was completely deficient in viral DNA synthesis at 39.5 degrees C. Comparative polyacrylamide gel electrophoresis of the ts 1-8, ts 199 and wild-type viral-coded polypeptides and cellular proteins produced in vivo at 34 degrees C and 39.5 degrees C during various periods post infection was performed. The results indicated that ts 1-8 and ts 199 were temperature-sensitive for the secondary suppression of host polypeptide synthesis. Production of the beta (early) and gamma (late) viral polypeptides was slightly delayed in the mutant-infected cells at early times post infection at both 34 degrees C and 39.5 degrees C. This delayed protein production was not evident at later times post-infection. The ts 1-8 and ts 199 mutants were distinct from the HSV-1 viron-associated host shutoff (vhs) mutants of Read and Frenkel (J. Virol. 46 (1983) 498).

Differential replication of SV40 and polyoma DNAs in Chinese hamster ovary cells

We have investigated the ability of CHO cells to allow growth of papovaviruses by analyzing viral DNA replication after transfection using the calcium-phosphate co-precipitation technique. These analyses showed that when SV40-containing plasmids were introduced into CHO cells, viral DNA replicated to a level of approximately 1000 copies per T antigen-expressing cell, and neither late proteins nor virus progeny were produced. When polyoma (Py)-containing plasmids were transfected into CHO cells, a ten-fold higher level of Py DNA was present per T antigen-positive cell, and viral capsid proteins and progeny virus were detected, indicating that CHO cells are not equally restricted for all papovaviruses. Infection with intact virions was restricted in both cases. These results indicate that either SV40 or Py DNA introduced into CHO cells are able to express their early viral functions, and that different interactions of cellular proteins involved in the replication machinery with viral nucleic acids and proteins result in different levels of viral DNA synthesis and virus progeny production. We propose that, because of their favorable genetic characteristics, CHO cells should, therefore, provide a valuable experimental system for definition of the cellular contributions to papovavirus replication.

Expression of viral early functions in rat 3Y1 cells infected with human papovavirus BK.

A plaque morphology mutant (pm-522) of BK virus (BKV) with a small deletion at map unit 0.72 can readily transform rat 3Y1 cells, but wild-type BKV (wt-501) cannot. We examined the expression of the viral early functions in BKV (wt-501 or pm-522)-infected 3Y1 cells within a 2-week period after infection, before foci of transformed cells became detectable, to know how the difference between the two BKVs occurs. After a high-multiplicity infection, comparable amounts of free viral DNA (forms I and II) were found by Southern blotting analyses to persist in the nuclei of the cells infected with wt and pm BKVs. Whereas the proportion of T antigen-positive cells, as revealed by the indirect immunofluorescence method with complement, remained at a level of 60% in pm BKV infection, the level of T antigen-positive cells in wt BKV infection decreased from the initial 45% to 1% on day 9. The results obtained by the immunoprecipitation analyses of radiolabeled proteins from the infected cells were consistent with the immunofluorescence data. Viral early mRNA was detectable on day 2 and increased on day 9 in pm BKV infection, but in wt BKV infection, the low level of early mRNA detected on day 2 disappeared on day 9. Cell DNA synthesis and cell growth were enhanced more in pm BKV infection than in wt BKV infection. The low level of viral DNA synthesis that occurred in the infected rat cells was more prominent in pm BKV infection than in wt BKV infection. These data indicate that the expression of viral early functions continued much longer in pm BKV-infected rat cells than in wt BKV-infected rat cells, where the expression was probably repressed soon after infection. Continued T antigen production directed by the unintegrated viral genomes appears to be required for efficient transformation of rat cells by BKV.

Inhibition of herpes simplex virus replication by succinyl concanavalin A

Incubation of herpes simplex virus type 1-infected cells with succinyl-concanavalin A, a derivative of the jack bean lectin concanavalin A, resulted in the decreased production of virus. The mode of inhibition by the lectin was unclear. No effect was apparent on the level of viral DNA synthesis. However, incubation of infected cells with increasing concentrations of the lectin appeared to result in a decrease in the quantity of viral protein produced within the cell. A reduction in the virus titer of 57 to 64% was observed upon direct incubation of extracellular virus in the presence of 50 to 100 micrograms of succinyl-concanavalin A per ml.