Bombyx mori Nucleopolyhedrovirus Encodes a DNA-Binding Protein Capable of Destabilizing Duplex DNA

A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from Bombyx mori) infected with the B. mori nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"L33180","term_id":"3745835","term_text":"L33180"}}L33180). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3′→5′ exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.

Nucleopolyhedroviruses (NPVs) have large (80- to 180-kb) circular double-stranded DNA (dsDNA) genomes, which replicate in nuclei of infected cells. Despite the widespread use of NPVs for the expression of foreign genes and their potential for pest control, little is known about the mechanism of their replication and the properties of their replication factors. The most widely studied baculovirus, Autographa californica multicapsid NPV (AcMNPV), has the potential to encode about 150 proteins (3), including factors required for virus DNA replication. The products of nine viral genes (ie-1, ie-2, lef-1, lef-2, lef-3, dnahel, dnapol, p35, and lef-7 or pe-38) are necessary and sufficient for efficient replication of transfected plasmid DNAs containing a putative baculovirus replication origin (16, 22). It is likely that DNA polymerase and DNA helicase, which are encoded by the viral genes dnapol and dnahel, respectively (20, 35), form a core of the virus DNA replication machinery. The roles of other factors are less obvious. Single-stranded DNA binding (SSB) protein function was proposed for the protein LEF-3, which binds specifically single-stranded DNA (ssDNA) (10, 14). However, direct proof for the SSB function of LEF-3 in viral DNA replication is lacking. In addition, SSB function was also suggested for LEF-7 on the basis of its predicted amino acid sequence (22). It was recently demonstrated that LEF-1 forms a complex with LEF-2 and may serve as a DNA primase (9). The function of IE-1, IE-2, and PE-38 may result from their ability to activate in trans expression of other genes required for virus replication. The transactivator IE-1 may also participate in the initiation of DNA replication, due to its ability to bind putative replication origins (7, 13, 17, 33). P35 is an inhibitor of apoptosis and may not be involved directly in DNA replication. Its stimulatory effect in the transient-replication assay may result from inhibition of virus-induced apoptosis in cells transfected with the replication genes. Several genes required for DNA replication (six essential and three stimulatory) were also identified in the genome of Orgyia pseudotsugata NPV (1). Homology of these genes to those required for replication of AcMNPV suggests similar replication mechanisms for the two viruses. The genome organization of the Bombyx mori NPV (BmNPV) closely resembles that of AcMNPV. Nineteen homologs of the AcMNPV late expression factor genes (lef genes) were identified in BmNPV (12). At least three of these, ie-2, lef7, and p35, are not essential for virus DNA replication as demonstrated by deletion analysis (12). Because the daughter DNA molecules synthesized under control of the nine essential viral genes appear to be synthesized as concatemers (16, 22, 31, 32), factors required for maturation of nascent DNA and its further processing are still unknown. Although the nine AcMNPV factors were sufficient for efficient DNA replication in Sf cells, an additional viral gene, designated hcf-1, was essential for replication in TN-368 cells (21), indicating dependence of the transient assay on host cell-specific factors. Few proteins involved in NPV DNA replication have been purified from infected cells and characterized in cell-free systems. Among them are AcMNPV DNA polymerase (28, 37), BmNPV DNA polymerase (27), AcMNPV DNA helicase (19), and AcMNPV LEF-3 (10, 14). Isolation of other replication proteins of NPVs is still anticipated.In this report we describe the purification of a viral DNA-binding protein (designated DBP) from BmNPV-infected cells. DBP binds preferentially to ssDNA and is capable of unwinding duplex DNA. The BmNPV open reading frame (ORF) encoding DBP (dbp gene) is a homolog of AcMNPV ORF25, whose product has not been identified so far.