The contributions of DNA polymerases alpha, delta, and epsilon to SV40 and nuclear DNA syntheses were evaluated. Proteins were UV‐crosslinked to nascent DNA within replicating chromosomes and the photolabelled polymerases were immunopurified. Only DNA polymerases alpha and delta were detectably photolabelled by nascent SV40 DNA, whether synthesized in soluble viral chromatin or within nuclei isolated from SV40‐infected cells. In contrast, all three enzymes were photolabelled by the nascent cellular DNA. Mitogenic stimulation enhanced the photolabelling of the polymerases in the alpha>delta>epsilon order of preference. The data agree with the notion that DNA polymerases alpha and delta catalyse the principal DNA polymerisation reactions at the replication fork of SV40 and, perhaps, also of nuclear chromosomes. DNA polymerase epsilon, implicated by others as a cell‐cycle checkpoint regulator sensing DNA replication lesions, may be dispensable for replication of the small, fast propagating virus that subverts cell cycle controls.

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The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BHV-1) is abundantly expressed and alternatively spliced in trigeminal ganglia. A mutant BHV-1 strain that contains three stop codons at the beginning of LR open reading frame (ORF)-2 (LR mutant virus) does not express ORF-2 or an adjacent reading frame that lacks an initiating ATG (RF-C). Calves latently infected with wild-type (wt) BHV-1, but not with the LR mutant virus, reactivate from latency, indicating that proteins encoded by the LR gene regulate the latency-reactivation cycle. The LR gene also contains another large ORF (ORF-1) that is approximately 200 bp downstream of stop codons inserted at the N-terminus of ORF-2. To test whether the LR mutant virus can expresses ORF-1, the authors developed antiserum directed against ORF-1. The ORF-1 antiserum recognizes specific proteins in bovine cells productively infected with wt BHV-1. ORF-1 protein expression is reduced, but not blocked, when bovine cells are infected with the LR mutant virus. Confocal microscopy demonstrated ORF-1 is present in the cytoplasm and nucleus of productively infected cells, whereas RF-C or a fusion protein containing RF-C localizes to the cytoplasm. Trigeminal ganglia from calves latently infected with wt BHV-1 contain neurons specifically stained with the ORF-1 antiserum. These studies suggest ORF-1 expression may be important for the BHV-1 latency-reactivation cycle.

Aims: A novel integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay was established for detection of infectious hepatitis A virus (HAV). Methods and Results:The specificity of tagged RT-PCR was assessed using HAV genomic positive-strand RNA extracted from HAV virions as reference. Water samples artificially contaminated with infectious or formalininactivated HAV were subjected to integrated cell culture (ICC)/RT-PCR and ICC/strand-specific RT-PCR assays respectively. The tagged RT-PCR had high specificity for HAV negative-strand RNA. By demonstrating the formation of negative-strand RNA replicative intermediate, ICC/strand-specific RT-PCR can distinguish between infectious and non-infectious HAV. The described method detected infectious HAV at inoculation level of 10 0 TCID 50 per flask within 4 days. Conclusions: The ICC/strand-specific RT-PCR is a novel, rapid, sensitive and reliable method for detection of infectious HAV. Significance and Impact of the Study: Coupled with a suitable virus concentration and purification system, ICC/strand-specific RT-PCR will provide a novel and rapid method for detection of infectious HAV in clinical, environmental and food samples. This assay may be used as an alternative method to test the effective inactivation of inactivated virus vaccines. It may also be adapted to assess the efficacy of disinfection of HAV and enteric viruses in foods and water.

The catalytic polypeptide of human DNA polymerase delta was overexpressed in BSC-40 cells (African green monkey kidney cell line) using the vaccinia virus/pTM1 system. The recombinant human DNA polymerase delta was purified to homogeneity in two steps using an immunoaffinity column and a single-stranded DNA-cellulose column. Levels of expression were about 1% of soluble cytosolic protein. The recombinant catalytic subunit was fully active and exhibited enzymatic properties similar to that of the native two-subunit enzyme including the possession of an associated 3' to 5' exonuclease activity. Recombinant pol delta was stimulated by proliferating cell nuclear antigen (PCNA); however, the degree of stimulation was lower than that of the native human enzyme. Analysis of a double mutant of the catalytic subunit, H142R/F144S, showed that it had a greatly reduced sensitivity to PCNA, suggesting that the PCNA binding site of pol delta may be located in this region of the N terminus.

The immediate-early protein, bICP0, of Bovine herpesvirus 1 (BHV-1) transactivates viral promoters and stimulates productive infection. bICP0 is expressed constitutively during productive infection, as its gene contains an immediate-early and an early promoter. Like other ICP0 homologues encoded by members of the subfamily Alphaherpesvirinae, bICP0 contains a zinc RING finger located near its N terminus. Mutations that disrupt the bICP0 zinc RING finger impair its ability to activate transcription, stimulate productive infection, inhibit interferon-dependent transcription in certain cell types and regulate subnuclear localization. bICP0 also interacts with a cellular chromatin-remodelling enzyme, histone deacetylase 1 (HDAC1), and can relieve HDAC1-mediated transcriptional repression, suggesting that bICP0 inhibits silencing of the viral genome. In this study, it was shown that bICP0 interacted with the histone acetyltransferase p300 during productive infection and in transiently transfected cells. In addition, p300 enhanced BHV-1 productive infection and transactivated a late viral promoter (gC). In contrast, a CH3-domain deletion mutant of p300, which is a dominant-negative mutant, did not activate the gC promoter. bICP0 and p300 cooperated to activate the gC promoter, suggesting that there is a synergistic effect on promoter activation. As p300 can activate certain antiviral signalling pathways (for example, interferon), it was hypothesized that interactions between p300 and bICP0 may dampen the antiviral response following infection. INTRODUCTIONInfection of cattle with Bovine herpesvirus 1 (BHV-1) can result in conjunctivitis, pneumonia, genital disorders, abortions and 'shipping fever', an upper respiratory tract infection (Tikoo et al., 1995). BHV-1 infection of bovine cells leads to rapid cell death and an increase in apoptosis (Devireddy & Jones, 1999). Viral gene expression is regulated in three distinct temporal phases: immediate-early (IE), early (E) and late (L) (Jones, 2003). The bICP0 protein is encoded by IE transcription unit 1 (Wirth et al., 1992) and activates expression of all three classes of viral promoter (Everett, 2000). The bICP0 gene is expressed constitutively during productive infection, as it has an IE and an E promoter that are activated by bICP0 (Fig. 1a; Fraefel et al., 1994). bICP0 associates with histone deacetylase 1 (HDAC1) and, in quiescent cells, bICP0 relieves HDAC1-mediated repression of transcription (Zhang & Jones, 2001). The ability of bICP0 to interact with HDAC1 may stimulate viral gene expression. Construction of a mutant BHV-1 that does not express the bICP0 protein has demonstrated that bICP0 plays an important role in productive infection of cultured bovine cells (Geiser et al., 2005). The ICP0 homologues encoded by BHV-1 and Human herpesvirus 1 (herpes simplex virus type 1, HSV-1) contain a well-conserved C 3 HC 4 zinc RING finger near their respective N termini. Mutational analysis has demonstrated the importance of the C 3 HC 4 zinc RING-finger domain of bICP0...

DNA polymerase ␦ is a heterodimer consisting of a catalytic subunit of 125 kDa and a small subunit of 50 kDa (p50). We have overexpressed p50 in Escherichia coli and have characterized the recombinant protein. p50 was readily overexpressed using the pET vector as an insoluble protein. A procedure was developed for its purification and renaturation. Examination of the physicochemical properties of renatured p50 showed that it is a monomeric protein with an apparent molecular weight of 60,000, a Stokes radius of 34 Å, and a sedimentation coefficient of 4.1 S. Its physical properties were indistinguishable from p50 expressed as a soluble protein using the pTACTAC vector. Examination of the effects of recombinant p50 on the activity of DNA polymerase ␦ showed that p50 is able to slightly stimulate (about 2-fold) the activity of the recombinant 125-kDa catalytic subunit using poly(dA)⅐oligo(dT) as a template in the absence of proliferating cell nuclear antigen. In the presence of proliferating cell nulear antigen, activity is stimulated about 5-fold. Seven stable hybridoma cell lines were established that produced monoclonal antibodies against p50. One of these antibodies (13D5) inhibited the activity of calf thymus DNA polymerase ␦. This antibody, when coupled to a solid support, also was found to provide a method for the immunoafffinity purification of recombinant p50 and of DNA polymerase ␦ from calf thymus or HeLa extracts. Immunoprecipitation and enzyme-linked immunosorbent assays also confirmed that p50 interacts with the catalytic subunit of DNA polymerase ␦.DNA polymerase (pol) 1 ␦ is involved in both DNA replication (1) and DNA repair (2) in mammalian cells. Its activity is stimulated by PCNA, a DNA sliding clamp which provides the processivity required for its role in DNA replication (3, 4). In addition to PCNA, a number of other accessory proteins are required for the assembly of a functional replication complex at the leading strand of the replication fork. These include the multi-subunit replication factor C (RF-C) complex and replication protein A (RP-A), a single-stranded DNA-binding protein (5). pol ␦ isolated from calf thymus (6) and human placenta (7) is a heterodimer of 125-and 50-kDa polypeptides. The catalytic activity had been clearly demonstrated to be associated with the 125-kDa subunit (7), and preparations of mammalian pol ␦ have been reported that contain only the active 125-kDa subunit (8, 9). Goulian et al. (9) isolated two preparations of mouse DNA polymerase ␦, one of which consisted of a single polypeptide of 123-125 kDa and was unresponsive to PCNA stimulation. It was suggested that the 50-kDa polypeptide is required for the stimulation of DNA polymerase ␦ by PCNA (9). However, yeast pol ␦ catalytic subunit overexpressed in Escherichia coli was stimulated 2.5-to 3-fold (10). Human pol ␦ catalytic subunit expressed in the vaccinia virus system also showed a slight increase in activity in the presence of PCNA (11). This increase in activity is significantly lower than the 34-fold stimul...