Characterization of <Emphasis Type="Italic">p</Emphasis>24 Gene of <Emphasis Type="Italic">Spodoptera litura</Emphasis> Multicapsid Nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) p24 gene is 753 bp long, potentially encoding 244 amino acids with a predicted molecular weight of 27.3 kDa. Homology analysis indicated that SpltMNPV P24 has 20–36% amino acid identity with that of other known baculoviruses. RT-PCR results showed that the p24 gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (ATAAG). Western blot analysis of extracts from SpltMNPV-infected S. litura cells detected a specific 28 kDa protein, and this protein was not N-glycosylated. Structural localization revealed that SpltMNPV P24 was associated with the nucleocapsid of occlusion-derived virus (ODV) as a complex form of 83 kDa.     

Identification and characterization of odv-e25 of Spodoptera litura multicapsid nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) odv-e25 is 684 bp long, potentially encoding 227 amino acids with a predicted molecular weight of 24.9 kDa. Homology analysis indicated that SpltMNPV ODV-E25 has 35–65% amino acid identity with that of other known baculoviruses. RT-PCR results revealed that the odv-e25 is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (TTAAG). Western blot analysis of odv-e25 expression with an antiserum made against 6 × His tagged ODV-E25 expressed in Escherichia coli indicated that it was present as a doublet of approximately 27 kDa from 24 h through 96 h in SpltMNPV-infected Spli-221 cells. Similar results were seen on Western blots of Spodoptera exigua (Se)MNPV-infected Se301 cells. Immunofluorescence analysis showed that ODV-E25 was predominantly present in the cytoplasm of SpltMNPV-infected cells and localized to the envelopes of occlusion-derived virus.     

Generation of a Recombinant Anticarsia Gemmatalis Multicapsid Nucleopolyhedrovirus Expressing a Foreign Gene under the Control of a Very Late Promoter

A recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) expressing -galactosidase under the control of the polyhedrin promoter was generated in our laboratory. To this end, we cloned the AgMNPV-2D genomic DNA fragment containing the polh gene and subcloned and sequenced the polyhedrin gene and its flanking regions. Based on this sequence information, sets of primers were designed to amplify the flanking regions by PCR, including appropriate restriction sites. The transfer vector (pAgPHZ) was constructed by the consecutive cloning of these PCR fragments flanking the Escherichia coli LacZ gene, in place of the polh gene. pAgPHZ was used for cotransfection of UFL-AG-286 insect cells with AgMNPV-2D DNA and the required recombinant, generated by homologous recombination with the polh locus, was identified by its polh ^–/LacZ ⁺ plaque phenotype. Its genome structure was confirmed by PCR, restriction digestion and Southern blot analyses. The kinetics and levels of expression of -galactosidase in UFL-AG-286 cells infected with the recombinant were tested by SDS-PAGE and enzymatic activity assays.     

Identification of the Apoptosis Inhibitor Gene <Emphasis Type="Italic">p49</Emphasis> of <Emphasis Type="Italic">Spodoptera litura</Emphasis> Multicapsid Nucleopolyhedrovirus

Baculoviruses possess two types of genes that suppressed apoptosis, p35 and inhibitor of apoptosis (iap). Computer-assisted analysis indicated that Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) ORF55 (designated as the p49 gene) display 79 and 31% amino acid identity with Spodoptera littoralis (Spli)MNPV P49 and Autographa californica (Ac)MNPV P35, respectively, Splt MNPV putative P49 contains a peptide cleavage site TVTDG recognized by death caspases. In marker rescue assay, Splt-p49 was able to suppress apoptosis induced by infection of a mutant AcMNPV deficient in p35 and rescue the mutant virus replication from apoptosis in Sf-9 cells.     

Characterization of the Polyhedrin Gene of Spodoptera litura Nuclear Polyhedrosis Virus

The polyhedrin gene (polh) of a characteristically distinct Spodoptera litura nuclear polyhedrosis virus isolate (SIMNPV) is identified in the HindIII-F fragment of the viral DNA. The nucleotide sequence of the 1057 base pair (bp) region of this fragment contains an open reading frame (ORF) without any intervening sequence for coding a polypeptide of 246 amino acids. Analysis of the nucleotide sequence and deduced amino acid sequence indicate that this has more than 70% sequence identity to known polyhedrins. The coding region is preceded by an AT rich region containing the conserved late promoter motif TAAG. The upstream promoter and coding regions of this polh gene are more similar to polh of the NPVs of Spodoptera frugiperda (Sf), Spodoptera exigua (Se) and Panolis flamea (Pf). This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and Classification of the Spodoptera littoralis Nucleopolyhedrovirus Inhibitor of Apoptosis Gene

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 (SlNPV). 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 SlNPV 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.     

The Lymantria dispar Nucleopolyhedrovirus Contains the Capsid-Associated p24 Protein Gene

During the course of investigations on a wild-type strain of Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV), a region of the viral genome was analyzed and found to contain 697 bp that is lacking in the sequenced strain (5–6) of LdMNPV (Kuzio et al., Virology 253, 17–34, 1999). The sequenced strain of LdMNPV contains a mutation in the 25 K few polyhedra (FP) gene, and exhibits the phenotype of a FP mutant. The additional sequence was located at approximately 81.4 map units within the viral genome, and was found in 10 different wild-type LdMNPV genotypic variants analyzed. Since the additional sequence was found in all wild-type virus strains analyzed, this sequence should be included in the representative LdMNPV genome. Sequence analysis of the genomic region containing the additional sequences revealed the presence of a homologue of the Autographa californica MNPV capsid-associated p24 gene (ORF 129). This gene, absent in LdMNPV isolate 5–6, is also present in the Orgyia pseudotsugata MNPV, Bombyx mori NPV, Spodoptera exigua MNPV, S. litura MNPV, Mamestra configurata MNPV, Helicoverpa armigera SNPV, H. zea SNPV, Buzura suppressaria SNPV, Xestia c-nigrum granulovirus, Plutella xylostella GV, and Cydia pomonella GV.     

Genomic Localization and Nucleotide Sequence of a lef-8 Gene of the Spodoptera Littoralis Nucleopolyhedrovirus

A gene similar to lef-8 of the Autographa californica (Ac) nucleopolyhedrovirus (MNPV) was identified in the Spodoptera littoralis (Spli) MNPV. The SpliMNPV lef-8-like gene was localized on the genomic map between 26.9 and 29 map units and is flanked by a chitinase gene and p47 gene. This gene arrangement differs from that of similar genes in the AcMNPV genome, where the lef-8 gene is located about 62 kbp from the chitinase gene and about 7 kbp from the p47 gene. Sequence analyses of the lef-8 gene revealed an ORF of 2730 nucleotides, predicted to encode a protein with M _r 104876. The putative protein is 60.9% identical to the AcMNPV LEF-8 and contains an identical sequence of a conserved motif of DNA-dependent RNA polymerases. Sequences downstream of the lef-8 gene contain two sequence repeats which resemble a repeated motif of the SpliMNPV enhancer element and other repetitive sequences from the viral genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physical and Partial Genetic Map of <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> Nucleopolyhedrovirus (SfMNPV) Genome

A Nicaraguan isolate of Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) is undergoing field trials for control of this pest in the Americas. This isolate is composed of multiple genotypes, some of which are deletion mutants. Identification of the genetic changes in deleted genotypes cannot be accomplished without the construction of a detailed physical map. In the present study, combinations of restriction endonuclease analysis and Southern blot analysis was performed. This map was refined by sequencing the termini of cloned restriction fragments. The SfMNPV genome was estimated to be 129.3 kb, 8 kb larger than the previously characterized Sf-2 variant from the United States, due to a deletion between 14.8 and 21.0 m.u. in the physical map described in this study. A total of 27.92 kb were sequenced, which represented 21.5% of the whole genome and included 38 ORFs. Comparison with other sequenced baculoviruses revealed that SfMNPV displayed the highest sequence identity (66%) and gene arrangement (78%) with Spodoptera exigua MNPV, sharing 36 putative ORFs. In addition, the genome organization was similar to that of SeMNPV, with minor differences. Phylogenetic analysis confirmed the close relatedness between SeMNPV and SfMNPV, suggesting they evolved from a common ancestor.     

Identification of Insertion and Deletion Genes in Autographa californica Nucleopolyhedrovirus Variants Isolated from Galleria mellonella,Spodoptera exigua,Spodoptera litura and Xestia c-Nigrum

The genomic DNA of four Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) variants isolated from Galleria mellonella, Spodoptera exigua, Spodoptera litura and Xestia c-nigrum was analyzed in comparison with the AcMNPV E2 strain. Restriction endonuclease analysis revealed a deletion and an insertion in collinear regions of the four variants. Polymerase chain reaction analysis indicated that, in the four variants, the deletion occurred in the region corresponding to AcMNPV C6 ORF86 (pnk/pnl). Also the insertion, with a length of approximately 1.1 kb, was commonly identified in the fragments corresponding to the PstI-J fragment (18.5 m.u.–21.2 m.u.) of AcMNPV E2. Sequencing analysis of the variant from S. litura showed that the insertion contains an additional open reading frame encoding 322 amino acids between homologues of AcMNPV ORF30 and ORF31 (the superoxide dismutase gene). This ORF has 82.8% amino acid identity to Bombyx mori NPV T3 ORF 22 (bro-a, one of the baculovirus repeated ORFs) and thus, it was named Splt-bro-a. Southern blot hybridization study indicated that the other three variants also contain Splt-bro-a homologue. In addition, the labeled Splt-bro-a gene weakly hybridized to the PstI-D fragment (99.0 m.u.–8.0 m.u.) of AcMNPV E2. This fragment contains AcMNPV ORF2, a member of bro family. The signal was also observed on the corresponding fragment of the four variants. This result suggested that two bro genes are present in the four variants, although AcMNPV E2 and C6 are known to contain a single bro gene.