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...

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...

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase ␥ by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol ␥. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3-portion of a 71-mer template containing 40 dT residues at its 5-end as a binding site for mtSSB. DNA pol ␥ bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5-dT 40 -tail in the 18:71-mer primer-template reduced the binding of DNA polymerase ␥ and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 335 exonuclease of DNA polymerase ␥. The size of fragments protected from 335 exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.Single-stranded DNA-binding proteins (SSB) 1 comprise a group of proteins which bind preferentially and with high affinity to single-stranded DNA. SSB proteins are present in prokaryotic and eukaryotic cells and are assumed to be essential for DNA metabolism in all organisms. One of the best characterized prokaryotic SSBs, Escherichia coli SSB, is a stable homotetramer of subunits containing 177 amino acids (18.8 kDa). Both biochemical and genetic data indicate that E. coli SSB is involved in DNA replication, repair, and recombination (1). Depending on the ionic conditions this protein binds to DNA in multiple modes with an apparent binding site size varying from 35 to 56 or 65 nucleotides (2, 3). Eukaryotes contain two different families of nuclear and mitochondrial SSB proteins. Heterotrimeric nuclear SSBs (RPA) (subunits of about 70, 32, and 14 kDa) have been found in all eukaryotic cells examined. These proteins are essential for replication, are involved in recombination and repair, and interact specifically with other proteins involved in DNA metabolism although they have little sequence homology with prokaryotic SSBs (4).Mitochondrial SSBs have been isolated from Xenopus laevis oocytes (5), rat liver (6, 7), yeast (8), and Drosophila (9). Two Xenopus mtSSBs have been described. One of these, mtSSB-1 has been fully sequenced (10) and is represented in a fulllength cDNA clone (11). The second, mtSSB-2, has not been cloned and has been only partially sequenced. The first 80 residues of mtSSB-2 reveal 91% identity to mtSSB-1, which contains 129 amino acids (14.6 kDa). Both Xenopus (12) and ...

We have recently identified a DNA-binding protein (DBP) from the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) which can destabilize double-stranded DNA (V. S. Mikhailov, A. L. Mikhailova, M. Iwanaga, S. Gomi, and S. Maeda, J. Virol. 72:3107–3116, 1998). DBP was found to be an early gene product that was not present in budded or occlusion-derived virions. In order to characterize the localization of DBP during viral replication, BmNPV-infected BmN cells were examined by immunostaining and confocal microscopy with DBP antibodies. DBP first appeared as diffuse nuclear staining at 4 to 6 h postinfection (p.i.) and then localized to several specific foci within the nucleus at 6 to 8 h p.i. After the onset of viral DNA replication at around 8 h p.i., these foci began to enlarge and eventually occupied more than half of the nucleus by 14 h p.i. After the termination of viral DNA replication at about 20 h p.i., the DBP-stained regions appeared to break down into approximately 100 small foci within the nucleus. At 8 h p.i., the distribution of DBP as well as that of IE-1 or LEF-3 (two proteins involved in baculovirus DNA replication) overlapped well with that of DNA replication sites labeled with bromodeoxyuridine incorporation. Double-staining experiments with IE-1 and DBP or IE-1 and LEF-3 further confirmed that, between 8 and 14 h p.i., the distribution of IE-1 and LEF-3 overlapped with that of DBP. However, IE-1 localized to the specific foci prior to DBP or LEF-3 at 4 h p.i. In the presence of aphidicolin, an inhibitor of DNA synthesis, immature foci containing IE-1, LEF-3, and DBP were observed by 8 h p.i. However, the subsequent enlargement of these foci was completely suppressed, suggesting that the enlargement depended upon viral DNA replication. At 4 h p.i., the number of IE-1 foci correlated with the multiplicity of infection (MOI) between 0.4 and 10. At higher MOIs (e.g., 50), the number of foci plateaued at around 15. These results suggested that there are about 15 preexisting sites per nucleus which are associated with the initiation of viral DNA replication and assembly of viral DNA replication factories.

Eight members of the HSP/HSC70 family were identified in Spodoptera frugiperda Sf9 cells infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by 2D electrophoresis followed by mass spectrometry (MALDI/TOF) and a Mascot search. The family includes five HSP70s induced by AcMNPV-infection and three constitutive cognate HSC70s that remained abundant in infected cells. Confocal microscopy revealed dynamic changes in subcellular localization of HSP/HSC70s in the course of infection. At the early stages (4 to 10 hpi), a fraction of HSPs is localized in distinct speckles in cytoplasm. The speckles contained ubiquitinylated proteins suggesting that they may be aggresomes where proteins targeted by ubiquitin are sequestered or processed for proteolysis. S. frugiperda HSP90 was identified in the 2D gels by Western blotting. Its amount was unchanged during infection. A selective inhibitor of HSP90, 17-AAG, decreased the rate of viral DNA synthesis in infected cells suggesting a supportive role of HSP90 in virus replication.