Alkaline nuclease (AN) of the Autographa californica multiple-capsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133) was expressed in recombinant baculovirus as a His 6 -tagged fusion and purified by sequential chromatography on Ni-NTA-agarose, DEAE-Toyopearl, and heparin-Sepharose. At all stages of purification, AcMNPV AN was found to copurify with a 44-kDa polypeptide which was identified as the baculovirus single-stranded DNA (ssDNA)-binding (SSB) protein, LEF-3. Sedimentation analysis in glycerol gradients of highly purified samples suggested that AN and LEF-3 are associated in a complex (designated *AN/L3), predominantly as heterodimers, although oligomeric forms containing both proteins were evident. In reactions with single-or double-stranded 62-mer oligonucleotides that were labeled with 32 P at the 5 or 3 ends, *AN/L3 carried out exonucleolytic hydrolysis of both substrates exclusively in a 533 direction. Saturation of ssDNA with an excess of LEF-3 prior to the addition of *AN/L3 resulted in a marked decrease in the rate of ssDNA hydrolysis. This suggests that excess LEF-3 may protect ssDNA from digestion by a AN-LEF-3 complex, thus regulating its activity in infected cells. The association of baculovirus AN with the viral SSB LEF-3 and the 533 exonuclease activity of this complex suggests that AN and LEF-3 may participate in homologous recombination of the baculovirus genome in a manner similar to that of exonuclease (Red␣) and DNA-binding protein (Red␤) of the Red-mediated homologous recombination system of bacteriophage .

Although the Baculoviridae are a large and diverse family of viruses, they are united by a number of shared features that form the basis for their unique life cycle. These include the mechanism of cell entry, genome replication and processing, and late and very late gene transcription. In this review, the molecular systems that are conserved within the Baculoviridae and that are responsible these processes are described.

Baculoviruses serve as a stress factor that can activate both death-inducing and cytoprotective pathways in infected cells. In this report, induction of heat shock proteins (HSPs) of the 70-kDa family (HSP/HSC70) in Sf-9 cells after infection with AcMNPV was monitored by Western blot analysis. Two-dimensional electrophoresis in polyacrylamide gel revealed changes in the cellular pattern of HSP/HSC70s and synthesis of a new member of the HSP/HSC70 family in the infected cells. Although infection with AcMNPV moderately increased the HSP/HSC70 content in cells under standard conditions, the infection potentiated the response to heat shock boosting the HSP/HSC70s content in infected cells several-fold in comparison with uninfected cells. Addition of KNK437, a known inhibitor of inducible HSPs, decreased the rate of viral DNA synthesis in infected cells more than one order of magnitude and markedly suppressed the release of budded viruses indicating the importance of the heat shock response for baculovirus replication.

Baculoviruses contain double-stranded (ds), 1 circular, supercoiled DNA genomes of 100 -180 kbp and belong to the family Baculoviridae, which includes two genera, the granuloviruses and the nucleopolyhedroviruses (NPVs) (1, 2). One NPV, Autographa californica multiple nucleocapsid NPV (AcMNPV), is widely used as a model for analysis of baculovirus replication in infected cells and for the generation of recombinant viruses for the expression of foreign genes. Replication of baculovirus genomes proceeds in discrete replication factories in the nuclei of infected cells (3, 4) presumably via a rolling-circle intermediates (5, 6), although the mechanisms of initiation, elongation, processing, and maturation have not been determined. Six viral factors including a transactivator of early gene transcription (IE-1), DNA polymerase, DNA helicase, DNA primase (LEF-1), an accessory factor (LEF-2), and ssDNA-binding protein (LEF-3) are necessary, and several other factors (P35, IE-2, PE38, and LEF-7) are stimulatory for replication of plasmids in the transient replication assays (7-12). The plasmid DNA synthesized in the presence of the essential and stimulatory replication factors in the transient assays is present as concatemeric molecules (5, 13) indicating that other viral products may be required for processing of nascent genomes. Two viral proteins, very late expression factor 1 (VLF-1) and alkaline nuclease (AN), have been predicted to participate in maturation of baculovirus genomes (14, 15). VLF-1 is required for expression of very late genes (16, 17) but also plays an unknown role in replication (14). VLF-1 is a member of the integrase/resolvase family of proteins (16), and it interacts preferentially with DNA crosses (18). This suggests that VLF-1 may function in the processing of branched intermediates in replication and recombination. The AN involvement in maturation of baculovirus genomes was predicted on the basis of structural homology of this enzyme and alkaline nuclease of viruses from the family Herpesviridae. Baculovirus AN contains five domains homologous to conserved motifs found in AN of alphaherpesviruses, although three other conserved motifs of the herpesvirus enzyme are not detected in the AcMNPV AN which is ϳ1.5-fold smaller than the herpes simplex virus type 1 (HSV-1) homolog (15). Although HSV-1 AN is not essential for viral DNA synthesis (19), deletion of the gene encoding AN results in the accumulation of complex branched concatemeric genomes indicating that AN either cleaves or prevents the generation of these structures (20,21). Because complex concatemeric intermediates are likely produced in DNA replication of baculoviruses (5, 6, 13), baculovirus AN may be also involved in the resolution of replication intermediates and genome maturation (15).Computer analyses reveal that AN of baculoviruses and herpesviruses belongs to a protein family typified by bacteriophage exonuclease (22, 23), a toroidal trimeric enzyme (24) that produces single-stranded DNA overhangs that serve as intermediates in...