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Baculoviruses possess two types of genes that suppressed apoptosis, p35 and inhibitor of apoptosis (iap). In this study we report the isolation and identification of an inhibitor of apoptosis gene Sliap in the genome of the Spodoptera littoralis nucleopolyhedrovirus (SINPV). The Sliap sequence predicted a 15 kDa polypeptide with only one BIR domain and a RING finger, both motifs characteristic of the IAP family of proteins, and a third specific acidic-rich motif. These characteristics, shared with the Spodoptera littura NPV IAP2/3, Epiphyas postvittana NPV IAP4, Lymantria dispar NPV IAP and Orgyia pseudotsugata NPV IAP4 (Orf 107) allowed us to classify them in a new homology group (IAP-4). Sliap was able to delay, but not to suppress, apoptosis induced by replication of a recombinant AcMNPV deficient in p35. In SINPV infected-SF9 cells Sliap was expressed earlier than sl-p49 suggesting that its role at the initiation of infection was to delay the apoptotic response of the host.

Spodoptera frugiperda SF9 cells infected with mutants of the Autographa californica nucleopolyhedrovirus (AcMNPV) which lack a functional p35 gene undergo apoptosis, aborting the viral infection. The Spodoptera littoralis nucleopolyhedrovirus (SlNPV) was able to suppress apoptosis triggered by vDeltaP35K/pol+, an AcMNPV p35 null mutant. To identify the putative apoptotic suppressor gene of SlNPV, overlapping cosmid clones representing the entire SlNPV genome were individually cotransfected along with genomic DNA of vDeltaP35K/pol+. Using this complementation assay, we isolated a SlNPV DNA fragment that was able to rescue the vDeltaP35K/pol+ infection in SF9 cells. By further subcloning and rescue, we identified a novel SlNPV gene, Slp49. The Slp49 sequence predicted a 49-kDa polypeptide with about 48.8% identity to the AcMNPV apoptotic suppressor P35. SLP49 displays a potential recognition site, TVTDG, for cleavage by death caspases. Recombinant AcMNPVs deficient in p35 bearing the Slp49 gene did not induce apoptosis and showed successful productive infections in SF9 cells, indicating that Slp49 is a functional homologue of p35. A 1.5-kbp Slp49-specific transcript was identified in SF9 cells infected with SlNPV or with vAc496, a vDeltaP35K/pol+-recombinant bearing Slp49. The discovery of Slp49 contributes to the identification of important functional motifs conserved in p35-like apoptotic suppressors and to the future isolation of p35-like genes from other baculoviruses.

A local strain of Helicoverpa armigera baculovirus was isolated from infected H. armigera larvae. Infectivity to Helicoverpa cells, restriction enzyme analysis and electron microscopy allowed its identification as a single embedded nucleopolyhedrovirus, designated HaSNPV-IS. Analysis of DNA replication, protein synthesis and polyhedrin expression in HaSNPV-infected cells located the late and very late phases of the viral cycle at 24 and 48 h after infection, respectively. The viral polyhedrin gene was isolated and characterized. It encoded for a polypeptide of 246 amino acid residues. A 32 kDa polypeptide was identified by immunoblot analysis using antipolyhedrin antiserum. The HaSNPV-IS polyhedrin DNA sequence revealed 99.4% of homology to the HzSNPV polyhedrin. The availability of this efficient replication system and the above knowledge paves the way to future genetic engineering of the HaSNPV.

Apoptosis was postulated as the main barrier to replication of the Autographa californica nuclear polyhedrosis virus (AcMNPV) in a Spodoptera littoralis SL2 cell line (N. Chejanovsky and E. Gershburg, Virology 209:519-525, 1995). Thus, we hypothesized that the viral apoptotic suppressor gene p35 is either poorly expressed or nonfunctional in AcMNPV-infected SL2 cells. These questions were addressed by first determining the steady-state levels of the p35 product, P35, in AcMNPV-infected SL2 cells. Indeed, very low levels of P35 were found in infected SL2 cells in comparison with those in SF9 cells. Overexpression of p35, in transient-transfection and recombinant-virus infection experiments, inhibited actinomycin D- and AcMNPV-induced apoptosis, as determined by reduced cell blebbing and release of oligonucleosomes and increased cell viability of SL2. However, SL2 budded-virus (BV) titers of a recombinant AcMNPV which highly expressed p35 did not improve significantly. Also, injection of S. littoralis larvae with recombinant and wild-type AcMNPV BVs showed similar 50% lethal doses. These data suggest that apoptosis is not the only impediment to AcMNPV replication in these nonpermissive S. littoralis cells, and probably in S. littoralis larvae, so p35 may not be the only host range determinant in this system.

The adeno-associated virus (AAV) rep gene proteins, Rep78 and Rep68, are required for replication of AAV DNA and bind to the AAV replication origin. An AAV genome having a Lys340 to His (K340H) mutation in the consensus purine nucleotide binding site of the rep gene protein exhibited a dominant-negative phenotype for DNA replication. We synthesized both wild-type and the K340H mutant Rep78 protein in a baculovirus expression system. Nuclear extracts of Sf9 cells containing these proteins were examined in gel mobility-shift assays with radiolabeled AAV terminal repeat DNA. Each protein bound specifically to the hairpin configuration of the AAV terminal repeat DNA to yield three shifted components. However the mobility of these components observed with the mutant Rep protein was slightly decreased compared to that with the wild-type Rep78. The addition of an antibody made against an oligopeptide from the carboxyl terminal region of the Rep78 protein generated novel shifted bands in the presence of either extract. Similar results were observed when the wild-type and mutant Rep proteins were expressed from an inducible expression system employing the human immunodeficiency virus type 1 transcription promoter in human 293 cells. These results suggest that the dominant-negative phenotype of the K340H mutation may be mediated by binding of the mutant protein to the AAV replication origin.

Adeno-associated virus (AAV) contains a multifunctional nonstructural gene, rep, which is required for AAV DNA replication and has pleiotropic effects on positive and negative regulation of gene expression. All of the parvovirus nonstructural genes contain a region of highly conserved amino acid homology. Within this conserved region is the consensus sequence for a purine nucleotide binding site. We constructed a mutant AAV having a mutation in this site by converting lysine 340 to histidine. The resulting mutant AAV genome, pNTC23, overproduced the mutant Rep proteins, indicating that these proteins are autoregulated. Furthermore, the mutant gene was unable to replicate but was able to inhibit in trans wild-type AAV DNA replication. Thus, pNTC23 represents a dominant negative mutant of AAV. These results suggest that rep has separate functional domains important for DNA replication.

The adeno-associated virus (AAV) rep gene is transcribed from two promoters, p5 and p19, which code for two over-lapping families of rep proteins. The proteins coded by p5 transcripts contain an amino-terminal domain not present in the proteins coded by p19 transcripts. The rep gene is required for AAV DNA replication and also mediates pleiotropic effects in positive and negative regulation of expression of genes driven by either AAV or heterologous promoters. All three functions require rep proteins coded by p5 transcripts. The functions of the rep proteins coded by the p19 transcripts could not be independently discerned since the coding region for these proteins and the p19 promoter are embedded within the coding region of the p5 transcript unit. We describe here an AAV mutant in which the putative AUG initiation codon in the p19 transcripts was altered and which did not express p19-coded rep proteins. This mutant exhibited normal AAV duplex RF DNA replication but was deficient in accumulation of AAV single-stranded progeny DNA and infectious AAV particles. This mutant defines a novel phenotype for a rep gene mutation and suggests a role for the rep proteins in the generation or accumulation of the viral SS DNA. Moreover this mutant distinguishes two different functions of the rep protein(s) in accumulation of the RF molecules and accumulation or processing of the SS DNA molecules, respectively.