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 Bo mbyx mori) infected with the B. mori nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achrom obacter protease I were partially or completely sequenced. The determi ned amino acid sequences indicated that DBP was encoded by an open rea ding frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the B mNPV genome (GenBank accession no. L33180). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose pr oduct has not been identified. BmNPV DBP is predicted to contain 317 a mino acids (calculated molecular mass of 36.7 kDa) and to have an isoe lectric 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 siz e for DBP is about 30 nt per protein monomer. DBP, but hot BmNPV LEF-3 , was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction th at DBP functions as a single-stranded DNA binding protein in virus rep lication.