Absence Of Viral DNA Replication Research Articles

Mutant Cellular AP-1 Proteins Promote Expression of a Subset of Epstein-Barr Virus Late Genes in the Absence of Lytic Viral DNA Replication.

Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator.IMPORTANCE Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.

TATA-binding protein and TBP-associated factors during herpes simplex virus type 1 infection: Localization at viral DNA replication sites

Host RNA polymerase II (RNAP II) is responsible for viral transcription of the herpes simplex virus type 1 (HSV-1) genome and is relocalized to viral DNA replication compartments. Thus, we investigated whether TATA-binding protein (TBP) and TBP-associated factors (TAFs) are recruited to sites of viral transcription and replication and whether TBP/TAF expressions are influenced upon infection. The protein levels of TBP, hsTAF1/TAF II250, hsTAF4/TAF II135, and hsTAF5/TAF II100 were constant during the early phase of infection and started to decrease late during infection. Only for hsTAF7/TAF II55 we sometimes observed a decrease already at 4–8 h postinfection (p.i.). Concomitantly with the relocalization of RNAP II, TBP and hsTAFs were redistributed to sites of viral DNA replication and transcription. In the absence of viral DNA replication TBP/hsTAFs were present in distinct nuclear dots, however, enlargement of the nuclear structures did not take place. Our results show that HSV-1 infection has no influence on the protein levels of TFIID components and leads to a redistribution of TBP and hsTAFs to prereplicative sites that enlarge to viral DNA replication compartments.

Relationship of herpes simplex virus genome configuration to productive and persistent infections.

Infection of susceptible cells by herpes simplex virus (HSV) can lead to productive infection or to latency, where the genomes persist in the nuclei of peripheral neurons in a quiescent state. Using the HSV strain d109, which does not express any viral genes and thus establishes a quiescent state in most cells, we observed that a fraction of genomes circularized upon infection. The expression of infected cell protein (ICP) 0, which is known to be involved in reactivation from latency and the promotion of productive infection, inhibited the formation of circular genomes. Circular genomes were not observed upon infection of fully permissive cells by wild-type virus, in either the presence or absence of viral DNA replication. However, productive infection in the absence of ICP0 resulted in the accumulation of a subpopulation of circular genomes. The proportion of circular genomes formed during infection with an ICP0 mutant was greater at low multiplicity of infection, a condition in which ICP0 mutants replicate poorly. In the complete absence of viral gene expression, it was found that only circular genomes persisted in cells. These results suggest that circularization of the HSV genome may not occur early in the productive phase of wild-type HSV infection, but rather during establishment of a quiescent state or latency, providing a possible strategy for long-term persistence. Additionally, the circularization and possible fate of HSV genomes are regulated by an activity of ICP0.

Relationships between polydnavirus genomes and viral gene expression

Polydnavirus genomes and viral gene functions are atypical for viruses. Polydnaviruses are the only group of viruses with segmented DNA genomes and have an unusual obligate mutualistic association with parasitic Hymenoptera, in which the virus is required for survival of the wasp host and vice versa. The virus replicates asymptomatically in the wasp host but severely disrupts lepidopteran host physiology in the absence of viral DNA replication. It is not surprising then that viral gene expression is divergent in its two insect hosts and that differences in viral gene expression are linked to these divergent functions. Some viral genes are expressed only in the wasp host while other viral genes are expressed only in the lepidopteran host and are presumed to be involved in the disruption of host physiological systems. Our laboratory has described the expression and regulation of a family of viral genes implicated in suppressing the lepidopteran immune system, the cys-motif genes. In conjunction with these studies we have described the physical organization of additional viral gene segments. We have cloned, mapped and begun the sequence analysis of selected viral DNA segments. We have noted that some viral DNA segments are nested and that nested viral DNA segments encode the abundantly expressed, secreted cys-motif genes. Conversely, other viral segments are not nested, encode less abundantly expressed genes and may be targeted intra-cellularly. These results suggest that nesting of segments in polydnavirus genomes may be linked to the levels of gene expression. By extension, the unique, segmented organization of polydnavirus genomes may be associated, in part, with the requirement for divergent levels of viral gene expression in lepidopteran hosts in the absence of viral DNA replication.

Simian virus 40 as a vector: recombinant viruses expressing individual polyoma T antigens

We constructed simian virus 40 (SV40)/polyomavirus recombinants by replacing in SV40 the T antigen coding region with polyoma early region sequences, either cDNAs encoding small, middle or large T antigen or the wild-type sequence coding all three proteins. The recombinants maintained the SV40 late region and origin of replication and were propagated in COS cells yielding recombinant virus preparations with titers of 10 6–10 7 infectious particles per milliliter. These viruses were characterized in productive infections of COS cells by analyzing early and late mRNA levels and by following synthesis of polyoma early proteins. In the absence of viral DNA replication, i.e. in infected monkey or mouse cells, expression of the polyoma T antigens was weak. Further experiments indicated that this was mostly due to high genomic instability during amplification, to lower levels of cDNA transcripts as compared to spliced mRNA, and possibly also to lower infectivity of the recombinant virions. It remains to be determined, whether these handicaps are unique to SV40/polyoma recombinants or whether SV40 is in general inadequate as a viral vector.

Characterization of nuclear structures in cells infected with herpes simplex virus type 1 in the absence of viral DNA replication.

Herpes simplex virus type 1 DNA replication occurs in nuclear domains termed replication compartments, which are areas of viral single-stranded DNA-binding protein (UL29) localization (M.P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868). In the presence of herpesvirus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-primase complex proteins containing short peptide epitopes which can be detected in an immunofluorescence assay, we have found that the helicase-primase complex localizes to prereplicative sites and replication compartments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have studied UL29 localization in cells infected with replication-defective viruses. Cells infected with viruses that fail to express one of the three helicase-primase subunits or the origin-binding protein show a diffuse nuclear staining for UL29. However, in the presence of polymerase inhibitors, mutant-infected cells contain UL29 in prereplicative sites. Replication-defective viruses containing subtle mutations in the helicase or origin-binding proteins behaved identically to their null mutant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites in the absence and presence of polymerase inhibitors. We propose that active viral polymerase prevents the formation of prereplicative sites. Models of the requirement of essential viral replication proteins in the assembly of prereplicative sites are presented.

The product of the UL6 gene of herpes simplex virus type 1 is associated with virus capsids

We report on the analysis of the UL6 and UL7 open reading frames of the herpes simplex virus type 1 (HSV-1) genome. The UL6 and UL7 transcripts were identified in HSV-1-infected cells by Northern blotting and shown to be coterminal at their 3′ ends. Both transcripts were synthesized in the presence of phosphonoacetic acid, although in reduced amounts, indicating that UL6 and UL7 are expressed as delayed-early or γ-1 genes. The 5′ ends of the two transcripts were mapped by S1 nuclease and primer extension analysis. A polyclonal antiserum directed against an Escherichia coli-expressed 6X His-UL6 fusion protein identified a protein of approximate M r 75,000 in cells infected with either HSV-1 or with a vaccinia virus recombinant expressing the HSV-1 UL6 protein. As with the transcript, the UL6 protein was synthesized at reduced levels in the absence of viral DNA replication. Western immunoblotting showed that the UL6 protein was present in purified virions but not in L-particles of HSV-1, and that it was located exclusively in the tegument/capsid fraction of virion. Further analysis of the UL6 protein revealed that this protein was associated with virus capsids.

Modification of the cascade model for regulation of vaccinia virus gene expression: purification of a prereplicative, late-stage-specific transcription factor

In vivo and in vitro studies have provided evidence that vaccinia virus late gene transcription factors are intermediate gene products synthesized exclusively after DNA replication. Here, we describe an additional transcription factor (P3 factor) that stimulates late gene transcription between 10- and 40-fold but is made in the absence of viral DNA replication. P3 factor activity was not detected either in uninfected cells or in purified virions. A > 1,500-fold purification of P3 factor was achieved by column chromatography of cytoplasmic extracts prepared from cells infected with vaccinia virus in the presence of a DNA replication inhibitor. P3 factor was stage specific, since it could not substitute for early or intermediate transcription factors. Evidence that late stage-specific transcription factors are made both before and after DNA replication necessitates a modification of the cascade model for vaccinia virus gene regulation.

Mutational Analysis of the Herpes Simplex Virus Type 1 Giycoprotein E Promoter

A set of linker-scanning mutations was constructed in the herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promoter to define cis-acting regulatory elements common to late viral promoters. The gE promoter is a late viral promoter that has some activity in the absence of viral DNA replication and is representative of the γ 1 class of HSV-1 promoters. Each promoter mutation was inserted upstream of the Escherichia coli lacZ gene in a recombinant virus, and the relative activities of β-galactosidase expressed from individual recombinant viruses were compared. This analysis identified two sequence elements, a TATA element and an element at the start of transcription, that corresponded to similarly positioned elements previously identified in the gC and gH late (γ 2) HSV-1 promoters. Mutation of the initiation element reduced expression from this promoter during normal lytic infection, but had no appreciable effect on expression in the absence of viral DNA replication, suggesting a specific role in late gene expression. Analysis of expression from hybrid gE/gC promoters revealed that the TATA and the initiation elements of these two promoters were interchangeable and that expression from the gE promoter in the absence of viral DNA replication was due to regulatory elements upstream from TATA element.

Characterization of the large tegument protein (ICP1/2) of herpes simplex virus type 1

ICP1/2 (also designated VP1/2) is a 270-kDa structural protein of herpes simplex virus type 1 (HSV-1) which is located in the tegument region of the virion. In this report we describe the production of a polyclonal antiserum specific for ICP1/2 and the use of this antiserum to examine the synthesis, processing, and intracellular localization of the viral polypeptide. Pulse-labeling studies indicated that ICP1/2 is synthesized late during infection, being initially detectable between 8 and 9 hr postinfection with the rate of synthesis continuing to increase until 11 to 12 hr postinfection. Further studies on the expression of ICP1/2 in the presence or absence of viral DNA replication indicated that the synthesis of the polypeptide is absolutely dependent on viral DNA replication. These results suggest that ICP1/2 represents a γ 2 (true late) gene product. Additionally, we have performed experiments to determine if ICP1/2 is post-translationally modified in HSV-infected cells. These studies indicated that ICP1/2 is phosphorylated on serine residues; however, we found no evidence to suggest that the protein is glycosylated. Using subcellular fractionation and indirect immunofluorescence techniques, we have determined that ICP1 /2 is diffusely distributed throughout the nucleus and cytoplasm of HSV-infected cells with no specific compartmentalization of the polypeptide.

Activation of simian virus 40 transcription in vitro by T antigen.

Simian virus 40 is repressed when the viral early gene product large tumor antigen (TAg) binds to specific sites within the viral origin and DNA replication ensues. Late transcription is activated by TAg, even in the absence of viral DNA replication. We show here that TAg produced in human 293 cells can selectively activate Simian virus 40 transcription in a cell-free system. In the absence of DNA binding by TAg, early and late transcription are both activated, as they are in vivo, suggesting that the effect might be mediated by a cellular component(s) utilized by both the early and late promoters. When TAg binds to the viral origin of replication, early transcription is repressed but the late promoter activation is unaffected. Various preparations of TAg differed in their activities, with some able both to bind DNA and to activate transcription and others able to do only one or the other. Since these variations might be explained by variable amounts of associated factors that copurified with TAg, we asked whether a bacterially derived protein could regulate transcription. An NH2-terminal 272-amino-acid fragment of TAg, produced in Escherichia coli as a glutathione S-transferase fusion protein, retains the ability to activate transcription in vitro, similar to that of the full-length protein. Structural features of this region that might be important are discussed.

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.

Identification and expression of a human cytomegalovirus early glycoprotein

A human cytomegalovirus early gene which possesses three temporally regulated promoters is located in the large unique component of the viral genome between 0.054 and 0.064 map units (C.-P. Chang, C.L. Malone, and M.F. Stinski, J. Virol. 63:281-290, 1989). This gene contains a major open reading frame (ORF) located 233 bases downstream of the cap site of an early unspliced RNA. The major ORF predicts a polypeptide of 17 kilodaltons (kDa) which contains a glycoproteinlike signal and anchor domains as well as potential N-glycosylation sites. Antisera were prepared against synthetic peptides derived from amino acid sequences within the major ORF. The antisera detected a viral glycoprotein of 48 kDa in infected cells and recognized the in vitro-translated 17-kDa protein early-gene product. The viral glycoprotein, designated gp48, was modified by N-linked glycans and possibly O-linked glycans. The synthesis of gp48 occurred in the absence of viral DNA replication but accumulated to the highest levels at late times after infection. Since gp48 was found in the virion, it is considered an early structural glycoprotein.

Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication.

We have examined the activation of the SV40 late promoter by inserting the late promoter and the viral origin of replication into chloramphenicol acetyltransferase (CAT) transient expression vectors. Very little late promoter activity was detected in CV-1 cells, compared with high activity in COS cells, in which replication occurs due to endogenous T antigen. Nonreplicative counterparts of these plasmids, containing a mutated origin of replication, produced significantly more late promoter activity in COS cells than any of the plasmids in CV-1 cells. When plasmids were cotransfected into CV-1 cells with a plasmid that supplies T antigen, the nonreplicative plasmid displayed 30% of the activity of the replicative plasmid. Using mutant T antigens unable to replicate viral DNA, late promoter activation occurred only with mutant T antigens that retain DNA binding activity. These results demonstrate that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication.

DNA methyltransferase induced by frog virus 3.

Over 20% of the cytosine bases in frog virus 3 DNA are methylated at the 5-carbon position. To determine whether this high degree of methylation is the result of a virus-specific enzyme, we examined the kinetics of induction and the substrate specificity of a DNA methyltransferase from frog virus 3-infected fathead minnow cells. A novel DNA methyltransferase activity appeared in the cytoplasm of infected cells at 3 h postinfection. This activity was induced in the absence of viral DNA replication and was therefore probably an early viral enzyme. In contrast to the methyltransferase activity extracted from uninfected cell nuclei, the cytoplasmic enzyme showed a strong template preference for double-stranded over single-stranded and for unmethylated over hemimethylated DNA. The dinucleotide sequence dCpdG was a necessary and sufficient exogenous substrate for methylation in vitro. A mutant of frog virus 3, isolated as resistant to 5-azacytidine and having unmethylated virion DNA, did not induce cytoplasmic DNA methyltransferase, leading to the conclusion that this activity is coded for by the virus.

Characterization of a temperature-sensitive mutant of frog virus 3 defective in DNA replication

We have characterized the defect of a temperature-sensitive (ts) DNA − mutant (ts 6642) of frog virus 3 (FV 3). At the nonpermissive temperature (30°) ts 6642 synthesized <3% of the viral DNA that was synthesized at the permissive temperature (23°). When ts 6642-infected cells were incubated at 30° for 4.0 hr and then shifted to permissive temperature, viral DNA synthesis started immediately even when protein synthesis was inhibited at the time of shiftdown. This result implies that at 30°, ts 6642 synthesized all the proteins required for viral DNA replication but that one of these was nonfunctional at the nonpermissive temperature. Further characterization revealed that ts 6642 was probably defective in the initiation of DNA replication. This conclusion was based on the following data: When ts 6642-infected cells incubated at 23° for 4.0 hr were shifted to 30°, there was a gradual decrease in viral DNA synthesis. By 1 to 1.5 hr after the shiftup, viral DNA synthesis was completely inhibited. Analysis of the density of the DNA synthesized after a shiftup in the presence of BUdR and FUdR suggested that residual viral DNA synthesis represented chain elongation, and not initiation of new rounds of DNA replication. The defective protein was therefore involved in the initiation process. Both wild-type FV 3 (FV 3 +) and ts 6642 induced the synthesis of thymidine kinase and DNA polymerase at 30°. Therefore, neither of these enzymes was involved in the DNA replication defect of ts 6642. At the nonpermissive temperature, ts 6642 synthesized all the viral proteins that were detectable at the permissive temperature. However, synthesis of late proteins was delayed, and never reached wild-type levels. Furthermore, the rate of synthesis of late proteins at 30° became dependent upon the multiplicity of infection. These results reinforce our previous conclusion ( R. Goorha and A. Granoff, 1974, Virology 60, 237–250) that in FV3 +-infected cells late proteins (and by implication late mRNAs) were synthesized in the absence of viral DNA replication.

Intracellular distribution and phosphorylation of virus-induced polypeptides in frog virus 3-infected cells

Analysis of the intracellular localization of frog virus 3 (FV 3)-infected cell polypeptides (ICPs) showed that although the largest amount of newly synthesized proteins were recovered, at any time postinfection, in the cytoplasmic fraction, most of the early viral-induced ICPs were present in the nuclei and the relative molar ratio of ICPs 90, 42, 31 was higher in the nuclei than in the cytoplasm. After 5–8 hr late polypeptides species were found concentrated in the nucleus (ICPs 70, 63, 12). The absence of viral DNA replication did not prevent the appearance of ICPs in the nucleus. Under abortive conditions produced by the replacement of arginine by canavanine, only a restricted set of ICPs was induced (early polypeptides) and the nuclear concentrations of ICPs 31 and 42 was modified. Newly phosphorylated proteins in infected cells were predominantly found in the nucleus, most species being viral induced. Moreover, following the induction of protein kinase, the specific activity of this enzyme was about 50 times higher in the nuclei than in the cytoplasmic fraction. These results extend the evidence for specific steps of FV 3 replication in the host nucleus, emphasizing the importance of the nucleus as the predominant site of viral polypeptide phosphorylation.

Synthesis of a structural adenovirus polypeptide in the absence of viral DNA replication

HeLa cells were pulse labeled with [ 35S]methionine at different times after infection with adenovirus type 2 to follow the kinetics of viral polypeptide synthesis. Cell lysates were prepared and viral proteins were analyzed by immunoprecipitation with antisera directed against “early” polypeptides or selected structural proteins of the adenovirus particle. The results showed that polypeptide IX, a structural component of the virion, is synthesized earlier than other structural polypeptides. Polypeptide IX is further distinguished from other components of the virion in that it is synthesized in the presence of cytosine arabinoside, a drug which blocks viral DNA replication. Cycloheximide, another drug which blocks viral replication and the onset of “late” viral transcription, also fails to prevent the accumulation of the messenger RNA coding for polypeptide IX, although mRNAs corresponding to the other structural viral components remain undetectable. Size fractionation of infected cell RNA showed that, like its “late” counterpart, the “early” messenger RNA for polypeptide IX sediments at 9 S. These results indicate that the regulation of the gene for polypeptide IX differs from that of other genes for structural adenovirus polypeptides.

Some adenovirus DNA is associated with the DNA of permissive cells during productive or restricted growth

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Similar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency integration.

Isolation and characterization of adenovirus type 12 DNA binding proteins

Infection of African green monkey kidney cells with type 12 adenovirus results in the production of two single strand specific DNA binding proteins. The molecular weights of these proteins are 60,000 and 48,000. Both proteins are synthesized in the absence of viral DNA replication and neither protein appears to correspond to any polypeptide detected in mature adenovirus virions. Temperature sensitive mutants from three different early, DNA negative, complementation groups (tsA, tsB, and tsC) have been tested for the production of these proteins at permissive and nonpermissive temperatures. Mutants of the tsB and tsC classes produce both DNA binding proteins at 32 and at 40°. TsA mutants produce both proteins at 32° but neither DNA-binding protein can be detected when these mutants are grown at 40°. The properties of adenovirus DNA binding proteins produced by types 2, 5, and 12 adenoviruses are compared.