Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

Abstract

DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His(6)-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA and that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.

Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication

“…Viruses with DNA genomes also require SSBs for replication of their genomes, and several have been identified in such diverse eukaryotic virus families as Adenoviridae, Herpesviridae, and Poxviridae. LEF-3 shares properties with these other well-characterized SSBs: it binds ssDNA (15,45); it oligomerizes in solution (33,34); it interacts with the helicase P143 (7,45); it closely associates with the viral DNA polymerase and the potential initiator IE-1 on viral DNA (17); it localizes to the nucleus (1); it can both unwind DNA duplexes and assist in annealing cDNA strands (31).…”

Changes in gene expression in the permissive larval host lightbrown apple moth (Epiphyas postvittana, Tortricidae) in response to EppoNPV (Baculoviridae) infection

“…3). These include two genes encoding proteins involved in the preparation of DNA for transcription; single stranded DNA binding protein (Mikhailov et al. , 1998, 2008) and LEF‐3 (Mikhailov, 2000, 2006), and three genes encoding immediate‐early activators of viral transcription regulatory proteins, IE‐0, IE‐1 and IE‐2 (Kovacs et al.…”

Identification of protein-protein interactions of the occlusion-derived virus-associated proteins of Helicoverpa armigera nucleopolyhedrovirus

“…In addition, DNA-binding protein (DBP) has also been reported to be a replication factor for BmNPV (Mikhailov et al, 1998). The interactions among these replication-associated proteins have been investigated in several studies and the interactions of LEF1-LEF2 and LEF3-helicase (Evans et al, 1999) and the self-associations of LEF3 , IE1 (Olson et al, 2001;Rodems & Friesen, 1995), IE2 (Imai et al, 2000) and DBP (Mikhailov et al, 2008) have been reported. In addition, some interactions in which replication-associated proteins were …”