Sites Of Viral DNA Replication Research Articles

The pseudorabies virus VP22 homologue (UL49) is dispensable for virus growth in vitro and has no effect on virulence and neuronal spread in rodents.

The tegument of herpesvirus virions is a distinctive structure whose assembly and function are not well understood. The herpes simplex virus type 1 VP22 tegument protein encoded by the UL49 gene is conserved among the alphaherpesviruses. Using cell biology and viral genetics, we provide an initial characterization of the pseudorabies virus (PRV) VP22 homologue. We identified three isoforms of VP22 present in PRV-infected cells that can be resolved by polyacrylamide gel electrophoresis. The predominant form is not phosphorylated and is present in virions, while the other two species are phosphorylated and excluded from virions. VP22 localized to the nucleus by 6 h postinfection, as determined by immunofluorescence and cell fractionation. VP22 immunofluorescence in the nucleus was both diffuse and in punctate structures. The punctate nuclear localization was the most pronounced form of staining and did not localize exclusively to sites of viral DNA replication. Unexpectedly, a VP22 null mutant had no obvious phenotypes during tissue culture infections and was similar to the wild type in all respects. Moreover, the VP22 null mutant was as virulent and neuroinvasive as the wild-type virus after infection of the rodent eye and spread to the brain using both anterograde and retrograde neuronal circuits.

Interaction with the Epstein-Barr virus helicase targets Zta to DNA replication compartments.

Zta has a dual role in the Epstein-Barr virus (EBV) lytic cycle, acting as a key regulator of EBV lytic gene expression and also being essential for lytic viral DNA replication. Zta's replication function is mediated in part through interactions with the core viral replication proteins. We now show interaction between Zta and the helicase (BBLF4) and map the binding region to within amino acids (aa) 22 to 86 of the Zta activation domain. In immunofluorescence assays, green fluorescent protein (GFP)-tagged BBLF4 localized to the cytoplasm of transfected cells. Cotransfection of Zta resulted in translocation of BBLF4-GFP into the nucleus indicating interaction between these two proteins. However, Zta with a deletion of aa 24 to 86 was unable to mediate nuclear translocation of BBLF4-GFP. Results obtained with Zta variants carrying deletions across the aa 24 to 86 region indicated more than one contact site for BBLF4 within this domain, and this was reinforced by the behavior of the four-point mutant Zta (m22/26,74/75), which was severely impaired for BBLF4 interaction. Binding of BBLF4 to Zta was confirmed using GST affinity assays. In both cotransfection-replication assays and replication assays performed in EBV-positive P3HR1 cells, the Zta (m22/26,74/75) mutant was replication defective. In Zta-transfected D98-HR1 cells, replication compartments could be detected by immunofluorescence staining using anti-BMRF1 monoclonal antibody. Cells transfected with Zta variants that were defective for helicase binding still formed replication compartments, but Zta was excluded from these compartments. These experiments reveal a role for the Zta-helicase interaction in targeting Zta to sites of viral DNA replication.

Comparison of the Intranuclear Distributions of Herpes Simplex Virus Proteins Involved in Various Viral Functions

Herpesviral transcription, DNA synthesis, and capsid assembly occur within the infected cell nucleus. To further define the spatial relationship among these processes, we have examined the intranuclear distributions of viral DNA replication, gene regulatory, and capsid proteins using dual label immunofluorescence and confocal microscopy. We observed that several of the viral DNA replication proteins localize preferentially to punctate structures within replication compartments while the major transcriptional activator, ICP4, and the ICP27 regulatory protein show a more diffuse distribution within replication compartments. The viral proteins that show a punctate distribution in replication compartments redistribute from these compartments to prereplicative sites when viral DNA replication is inhibited, whereas viral proteins that show a diffuse distribution remain within replication compartments when viral DNA replication is inhibited. Thus the sites of viral DNA replication and late transcription appear to be distinct but codistribute within the boundaries of replication compartments. The major capsid protein, ICP5, also localizes initially to a diffuse distribution within replication compartments, but during the time of maximal progeny virus assembly, ICP5 becomes localized to punctate structures within replication compartments that are often near the punctate structures occupied by viral DNA replication proteins. Hence the processes of viral DNA replication, late transcription, and capsid assembly show a general overlapping distribution within replication compartments but appear to be located at distinct sites within these regions of the infected cell nucleus.

Adenovirus preterminal protein binds to the CAD enzyme at active sites of viral DNA replication on the nuclear matrix.

Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) via an interaction mediated by the precursor of the terminal protein (pTP). The identities of cellular proteins involved in these complexes have remained obscure. We present evidence that pTP binds to a multifunctional pyrimidine biosynthesis enzyme found at replication domains on the NM. Far-Western blotting identified proteins of 150 and 240 kDa that had pTP binding activity. Amino acid sequencing of the 150-kDa band revealed sequence identity to carbamyl phosphate synthetase I (CPS I) and a high degree of homology to the related trifunctional enzyme known as CAD (for carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting. Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant. Additionally, in vitro-translated epitope-tagged pTP and CAD were immunoprecipitable as a complex, indicating the occurrence of a protein-protein interaction. Confocal fluorescence microscopy of Ad-infected NM showed that pTP and CAD colocalized in nuclear foci. Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation. These data support the concept that the pTP-CAD interaction may allow anchorage of Ad replication complexes in the proximity of required cellular factors and may help to segregate replicated and unreplicated viral DNA.

Nuclear sites of herpes simplex virus type 1 DNA replication and transcription colocalize at early times postinfection and are largely distinct from RNA processing factors.

We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation.

Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells.

We have visualized the intranuclear topography of adenovirus replication and transcription in infected HeLa cells. The results show that viral DNA replication occurs in multiple foci that are highly organized in the nucleoplasm. Pulse-chase experiments indicate that newly synthesized viral double-stranded DNA molecules are displaced from the replication foci and spread throughout the nucleoplasm, while the single-stranded DNA replication intermediates accumulate in adjacent sites. Double-labelling experiments and confocal microscopy show that replication occurs in foci localized at the periphery of the sites where single-stranded DNA accumulates. The simultaneous visualization of viral replication and transcription reveals that the sites of transcription are predominantly separated from the sites of replication. Transcription is detected adjacent to the replication foci and extends around the sites of single-stranded DNA accumulation. These data indicate that newly synthesized double-stranded DNA molecules are displaced from the replication foci and spread in the surrounding nucleoplasm, where they are used as templates for transcription. Splicing snRNPs are shown to co-localize with the sites of transcription and to be excluded from the sites of replication. This provides evidence that splicing of viral RNAs occurs co-transcriptionally and that the sites of viral DNA replication are spatially distinct from the sites of RNA transcription and processing.

Nuclear factor I is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells.

During the S phase of the eukaryotic cell cycle and in virus-infected cells, DNA replication takes place at discrete sites in the nucleus, although it is not clear how the proteins involved in the replicative process are directed to these sites. Nuclear factor I is a cellular, sequence-specific DNA-binding protein utilized by adenovirus type 2 to facilitate the assembly of a nucleoprotein complex at the viral origin of DNA replication. Immunofluorescence experiments reveal that in uninfected cells, nuclear factor I is distributed evenly throughout the nucleus. However, after a cell is infected with adenovirus type 2, the distribution of nuclear factor I is dramatically altered, being colocalized with the viral DNA-binding protein in a limited number of subnuclear sites which bromodeoxyuridine pulse-labeling experiments have identified as sites of viral DNA replication. Experiments with adenovirus type 4, which does not require nuclear factor I for viral DNA replication, indicate that although the adenovirus type 4 DNA-binding protein is localized to discrete nuclear sites, this does not result in the redistribution of nuclear factor I. Localization of nuclear factor I to discrete subnuclear sites is therefore likely to represent a specific targeting event that reflects the requirement for nuclear factor I in adenovirus type 2 DNA replication.

Cooperation of EBV DNA Polymerase and EA-D(BMRFl) in vitro and colocalization in nuclei of infected cells

Expression of the Epstein-Barr virus (EBV) DNA polymerase (EBVpol) open reading frame (BALF5) by in vitro transcription-translation yielded a 116-kDa primary translation product. Enzymatic DNA polymerase activity of the in vitro translated polypeptide required the presence of the 47-kDa BMRF1 (EA-D) gene product. Antiserum raised to the BALF5 gene product expressed in Escherichia coli specifically precipitated a 116-kDa polypeptide in extracts of latently infected lymphoblastoid cells induced for EBV replication. Immunofluorescence microscopy revealed colocalization of the EBVpol and EA-D(BMRF1) to discrete foci within the nuclei of induced cells; however, the blockade of viral DNA synthesis resulted in diffuse nuclear staining patterns for both antigens. Bromodeoxyuridine staining of these discrete foci colocalizing with EBVpol suggests that they are sites of early viral DNA synthesis. These observations suggest that EA-D(BMRFl) may be an accessory protein of the EBV DNA polymerase which colocalizes in vivo with EBVpol to sites of viral DNA replication and cooperates in vitro to form an active EBVpol holoenzyme.

Pattern of polyomavirus replication from infection until tumor formation in the organs of athymic nu/nu mice

Inbred athymic nu/nu BALB/c mice were injected subcutaneously with the highly oncogenic polyomavirus A2 strain, and the sites of viral DNA replication were determined by whole mouse section hybridization (T. W. Dubensky, E. A. Murphy, and L. P. Villareal, J. Virol. 50:779-783, 1984) and Southern blot analysis. We show that infection is persistent in some epithelial tissues (skin, mammary, and salivary glands), in lymphoid organs (spleen and nodes), and in mesenchymal bone tissue. Only mammary glands and bones were targets for tumor formation. Although the same pattern of infection was observed in males and females, mammary adenocarcinomas were induced exclusively in females, while the frequency of osteosarcomas was similar in both sexes. No viral DNA or lytic lesion was detected in kidney, liver, or lung tissue. The restricted targeting of polyomavirus oncogenicity in nude mice, compared with newborn immunocompetent animals, inoculated via the same route with the same virus strain, therefore does not reflect selective tissue targeting of virus replication. These results further document the influence of the age, immunological status, and genetic background of the host on the pattern of viral infection and tumor formation.

Analysis by in Situ hybridization and autoradiography of sites of replication and storage of single- and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells

The distribution in the different compartments of infected nuclei of double-stranded (ds) and single-stranded (ss) adenovirus type 5 (Ad5) DNA and of the sites of viral DNA replication were examined on thin sections of Lowicryl-embedded material. The DNA is visualized with a biotinylated viral probe and immunogold labeling of biotin, and its replication is monitored by high-resolution autoradiography after short pulses with tritiated thymidine. The first detectable sites of viral DNA, named early replicative sites, contained all the ss and ds viral DNA and viral replicative activity. At a later stage of nuclear transformation, they gave rise to two new structures. The compact fibrillar ssDNA accumulation sites enlarged greatly and became transformed functionally to become a transient site of accumulation of large numbers of ss replicative intermediates. Double-stranded viral DNA and its replicative activity shifted primarily into immediately surrounding fibrillogranular peripheral replicative zones. Ad5 DNA replication continues in the ssDNA accumulation sites but it is intermittent, whereas in the peripheral replicative zones it is continuous. Still later in infection, a single, large, centrally located mass of dense fibrils, the viral genome storage site, developed in each nucleus which proved to be the main site of storage of nonreplicating, nonencapsidated, ds viral genomes. We discuss the possible distribution of the various viral DNA replicative intermediates among these virus-induced intranuclear structures.