Transient expression of the vaccinia virus DNA polymerase is an intrinsic feature of the early phase of infection and is unlinked to DNA replication and late gene expression

Abstract

We have studied the expression pattern of the vaccinia virus DNA polymerase during the viral replicative cycle. To monitor polymerase synthesis, a polyclonal antiserum was raised against a TrpE-DNA polymerase fusion protein. Immunoprecipitation and Sl analyses revealed that polymerase synthesis and mRNA levels peak by 2 to 3.5 h postinfection during wild-type infections and then decline, becoming barely detectable by 5 to 6.5 h postinfection. Blocking viral DNA replication by performing infections with temperature-sensitive DNA-mutants at the nonpermissive temperature or by performing wild-type infections in the presence of cytosine ,B-D-arabinofuranoside had no effect on polymerase expression. These results indicate that the transient expression of the DNA polymerase is regulated independently of intermediate and late viral gene expression. Cycloheximide, which inhibits protein synthesis and prevents secondary uncoating, caused prolonged and elevated levels of polymerase transcription. Early viral proteins and uncoating, rather than exhaustion of the encapsidated transcription machinery, are presumed to mediate the cessation of polymerase transcription. In the presence of aphidicolin, the polymerase transcripts were maintained at maximal levels rather than exhibiting their normal decline. This inhibition of RNA decay was seen even in infections performed with isolates encoding aphidicolin-resistant DNA polymerases, suggesting that aphidicolin may interfere directly with the process of RNA degradation. Under these conditions, polymerase synthesis remained transient and was not prolonged, despite the continuing presence of available mRNA. These observations suggest that early mRNAs may experience a loss in translation efficiency as infection progresses.

Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins

“…2A DNA replication and subsequent late gene expression fail to A-A 39.50C at 7 hpi occur. This finding is consistent with our observation that the transient synthesis of two other early replication proteins, E9 (DNA polymerase) and D5, is also independent of 0 the progression of infection to DNA replication and late gene //0 expression (11,29). There appears to be at least a subset of T t°early proteins whose synthesis is regulated independently of A the transition to DNA replication and later transcriptional T O/ stages, although initial studies of DNAmutants revealed…”

“…The fusion protein was then used as an antigen for the generation of a rabbit polyclonal antiserum. The presence of antibodies reactive to the B1 protein was assessed by immunoprecipitation of metabolically labeled extracts (22,29) and immunoblotting (41).…”

“…The lysates described above were thawed and micrbcentrifuged at 4°C for 20 min to remove cryoprecipitates. Four hundred microliters of lysate, prepared from 0.5 x 106 cells, was diluted with an additional 800 ,ul of phospholysis buffer, and incubated with 5 ,ul of preimmune or anti-Bl serum on ice for 4 h. The immune complexes were recovered with protein A-Sepharose as previously described (29). The antigen was liberated from the complex by boiling in protein sample buffer (1% SDS, 1% 2-mercaptoethanol, 50 mM Tris [pH 6.8], 10% glycerol) and then resolved by electrophoresis through SDS-12% polyacrylamide gels (26).…”

Cytoplasmic factories, virus assembly, and DNA replication kinetics collectively constrain the formation of poxvirus recombinants

“…Little EdU incorporation was detected at the latest time point (8 hr post-infection) although even then some ongoing DNA synthesis could still be detected in the factories at 5-10% of the levels detected in the 3-4 hr period. These kinetics parallel, but lag 1-2 hr behind the kinetics of E9L gene expression [32].…”

Vaccinia virus nucleoside triphosphate phosphohydrolase I controls early and late gene expression by regulating the rate of transcription

“…Immunoprecipitation. DNA polymerase immunoprecipitation was performed as previously described (34). For NPH-I pulse-chase experiments, BSC-40 and Ltk(-) confluent monolayers were infected with ts36 (MOI, 10) at 31°C for 6 h. Cultures were labeled at this temperature by incubation in methionine-free Dulbecco's modified medium supplemented with [135S]methionine at 100 iLCi/ml, and after 30 min, labeling was stopped by addition of unlabeled methionine.…”