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Purification and functional characterization of several essential DNA replication proteins from Autographa californica polyhedrosis virus
The Autographa californica polyhedrosis virus (ACMNPV) replicates in the nuclei of infected cells and encodes several proteins required for viral DNA replication. Transient replication assay of plasmids containing a viral origin have shown that at least six viral genes (dna pol, helicase, lef-1, lef-2, lef-3, and iel) are essential while products of the ie2, pe38, p35, and lej7 genes stimulate the level of DNA replication. To functionally characterize viral replication proteins, we purified single-stranded binding protein (SSB) from AcMNPV-infected insect cells using affinity chromatography. Control experiments using uninfected cell extracts suggested that the SSB was encoded by the virus. SSB bound to single-stranded DNA in solution, and competition binding experiments showed that SSB binds to ssDNA preferentially and that it binds to ssDNA in a non-sequence-specific manner. To determine whether SSB was encoded by the ACMNPV lef-3 gene, we cloned the lef-3 open reading frame under the control of the bacteriophage T7 promoter. Immunochemical analysis indicated that LEF-3 produced in bacteria or in rabbit reticulate lysates specifically reacted with monoclonal antibodies raised against purified SSB and biochemical analysis indicated purified LEF-3 from bacteria overexpression systems bound to single-stranded DNA in solution. In addition, temporal expression of LEF-3/SSB during virus infection was examined by inimunoblot analysis. The viral DNA polymerase was purified to near homogeneity from infected cell nuclear extracts using conventional chromatographic methods. Purified viral DNA polymerase had intrinsic 3'-5' exonuclease activity and was sensitive to several metabolic drugs. Template challenge experiments using singly-primed ssDNA templates indicated that the baculovirus DNA polymerase was highly processive. As initial steps to understand the functions of AcMNPV LEF-I and LEF-2 proteins, the genes were cloned into bacterial expression plasmids and purified to near homogeneity. Antisera specific for LEF-I and LEF-2 were prepared and used to study their temporal expression by immunoblotting analysis of cell extracts prepared at various times during virus infection. A modified ELISA assay was used to show that LEF-I and LEF-2 proteins can interact directly and specifically in vitro.
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Includes bibliographical references: pages 116-126.