The baculovirus replication factors LEF-1 and LEF-2 of the Autographa californica multinucleocapsid nucleopolyhedrovirus were overexpressed as fusions containing a hemagglutinin (HA) epitope and a HIS 6 tag using recombinant baculoviruses. LEF-1 was purified to near homogeneity and found to have primase activity in an indirect assay employing Escherichia coli DNA polymerase I (Klenow enzyme) and poly(dT) template. The LEF-1 primase products were also directly characterized by electrophoresis in 20% polyacrylamide-8 M urea gels and agarose gels. Primer synthesis was time dependent, and products of several hundred nucleotides or more were observed from the M13 single-stranded DNA (ssDNA) template. The LEF-1 primase was absolutely dependent on divalent cations (Mg 2؉ ), and optimal activity was supported by 10 mM MgCl 2 . An alkaline pH (8.8 to 9.4) was optimal, whereas monovalent salt (KCl) was inhibitory. Mutation of an invariant aspartic acid in a putative primase domain caused LEF-1 activity to be abolished. Upon ultracentrifugation in glycerol gradients, LEF-1 was found to have a sedimentation coefficient of 3S that is consistent with its being present as a monomer. Elution profiles of LEF-1 and LEF-2 from ssDNA-cellulose and DEAE resin suggested that LEF-2 may bind to both DNA and LEF-1.The Baculoviridae are a large and diverse family of rodshaped, enveloped, occluded viruses that are pathogenic for invertebrates, particularly members of the Insecta. They have been reported from over 600 species, most of which are members of the Lepidoptera, Diptera, and Hymenoptera (34). Two genera of baculoviruses have been characterized, and they include the nucleopolyhedroviruses (NPVs) (47), which have numerous virions within large polyhedron-shaped occlusion bodies, and the granuloviruses (52), which commonly have a single virion within small granular occlusion bodies. Baculovirus genomes consist of double-stranded, circular, supercoiled DNA of 100 to 180 kb, depending on the strain of virus (20). Although evidence suggests that baculovirus genomes may replicate via a rolling-circle intermediate (31,42), the mechanisms of initiation, elongation, processing, and maturation have not been determined.Baculovirus DNA replication has been shown to be associated with discrete replication factories in the nuclei of infected cells (41). In addition, a conserved set of genes that are essential or highly stimulatory for transient DNA replication have been identified for Autographa californica multinucleocapsid NPV (AcMNPV) (26, 33), Orgyia pseudotsugata MNPV (OpMNPV) (reviewed in reference 1), and Lymantria dispar MNPV (44). These include genes encoding a DNA polymerase homolog, a DNA helicase homolog, ie-1, a transactivator of early gene transcription, and late expression factors (LEFs) encoded by lef-1, -2, and -3. The DNA polymerase and DNA helicase homologs were subsequently shown to have activities associated with these enzymes (35,36). In addition, lef-3 encodes a product with the properties of a single-stranded DNA (SSB) bi...
