THE LEF-3 GENE OF AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS ENCODES A SINGLE-STRANDED-DNA BINDING-PROTEIN

The Autographa californica nuclear polyhedrosis virus (AcNPV) replicat es in the nuclei of infected cells and encodes several proteins requir ed for viral DNA replication. As a first step in the functional charac terization of viral replication proteins, we purified a single-strande d DNA-binding protein (SSB) from AcNPV-infected insect cells. Nuclear extracts were chromatographed on single-stranded DNA agarose columns. An abundant protein with an apparent molecular weight of 43,000 was el uted from the columns at 0.9 to 1.0 M NaCl. This protein was not evide nt in extracts prepared from control cells, suggesting that the SSB wa s encoded by the virus. SSB bound to single-stranded DNA in solution, and binding was nonspecific with respect to base sequence, as single-s tranded vector DNA competed as efficiently as single-stranded DNA cont aining the AcNPV origin of DNA replication. Competition binding experi ments indicated that SSB showed a preference for single-stranded DNA o ver double-stranded DNA. To determine whether SSB was encoded by the l ef-3 gene of AcNPV, the lef-3 open reading frame was cloned under the control of the bacteriophage T7 promoter. Immunochemical analyses indi cated that LEF-3 produced in bacteria or in rabbit reticulocyte lysate s specifically reacted with antiserum produced by immunization with pu rified SSB. Immunoblot analyses of infected cell extracts revealed tha t SSB/LEF-3 was detected by 4 h postinfection and accumulated through 48 h postinfection.