Abstract
BackgroundReplication of the vaccinia virus genome occurs in cytoplasmic factory areas and is dependent on the virus-encoded DNA polymerase and at least four additional viral proteins. DNA synthesis appears to start near the ends of the genome, but specific origin sequences have not been defined. Surprisingly, transfected circular DNA lacking specific viral sequences is also replicated in poxvirus-infected cells. Origin-independent plasmid replication depends on the viral DNA polymerase, but neither the number of additional viral proteins nor the site of replication has been determined.ResultsUsing a novel real-time polymerase chain reaction assay, we detected a >400-fold increase in newly replicated plasmid in cells infected with vaccinia virus. Studies with conditional lethal mutants of vaccinia virus indicated that each of the five proteins known to be required for viral genome replication was also required for plasmid replication. The intracellular site of replication was determined using a plasmid containing 256 repeats of the Escherichia coli lac operator and staining with an E. coli lac repressor-maltose binding fusion protein followed by an antibody to the maltose binding protein. The lac operator plasmid was localized in cytoplasmic viral factories delineated by DNA staining and binding of antibody to the viral uracil DNA glycosylase, an essential replication protein. In addition, replication of the lac operator plasmid was visualized continuously in living cells infected with a recombinant vaccinia virus that expresses the lac repressor fused to enhanced green fluorescent protein. Discrete cytoplasmic fluorescence was detected in cytoplasmic juxtanuclear sites at 6 h after infection and the area and intensity of fluorescence increased over the next several hours.ConclusionReplication of a circular plasmid lacking specific poxvirus DNA sequences mimics viral genome replication by occurring in cytoplasmic viral factories and requiring all five known viral replication proteins. Therefore, small plasmids may be used as surrogates for the large poxvirus genome to study trans-acting factors and mechanism of viral DNA replication.
Highlights
Replication of the vaccinia virus genome occurs in cytoplasmic factory areas and is dependent on the virus-encoded DNA polymerase and at least four additional viral proteins
Vaccinia virus (VAC), the prototype for the family Poxviridae, is a large double-stranded DNA virus that encodes numerous enzymes and factors needed for RNA and DNA synthesis, enabling it to replicate in the cytoplasm of infected cells [1]
Efforts to locate a specific origin of replication in the VAC genome led to the surprising conclusion that any circular DNA replicated as head-totail tandem arrays in cells infected with VAC [29,30]
Summary
Replication of the vaccinia virus genome occurs in cytoplasmic factory areas and is dependent on the virus-encoded DNA polymerase and at least four additional viral proteins. Transfected circular DNA lacking specific viral sequences is replicated in poxvirus-infected cells. Origin-independent plasmid replication depends on the viral DNA polymerase, but neither the number of additional viral proteins nor the site of replication has been determined. A model for poxvirus DNA replication begins with the introduction of a nick near one or both ends of the hairpin termini, followed by polymerization of nucleotides at the free 3'-OH end, strand displacement and concatemer resolution [25,26]. It was considered that plasmid replication might be initiating non-perhaps at random nicks in DNA
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