Identification of a novel occlusion derived virus-specific protein in Spodoptera exigua multicapsid nucleopolyhedrovirus.

Understanding the molecular basis of the distinct biological properties of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), such as its narrow host range and high virulence, requires detailed information on the temporal expression and subcellular localization of SeMNPV gene products. The expression of two unique SeMNPV ORFs, 116 (Se116) and 117 (Se117), which show 45% amino acid similarity, was analyzed. Se116 and Se117 were expressed both in cultured cells and in larvae of S. exigua as polyadenylated transcripts of 0.80 and 0.75 kb, respectively. These transcripts initiated from ATCA(G/T)T promoter motifs, commonly found for baculovirus early genes. Se116 transcripts were detected with increasing abundance from 8 to 48 h p.i., whereas Se117 transcripts were present from 4 h p.i. and most abundantly at 24 h p.i. Western blot analysis of infected Se301 cells revealed 27- and 23-kDa proteins for Se116 and Se117, respectively. C-terminal GFP-fusion proteins of Se116 and Se117 were primarily localized in the nucleus of Se301 cells. When Se301 cells were infected with SeMNPV, both GFP-fusion proteins were localized in the virogenic stroma of the nucleus. While the function of the Se116 protein is still enigmatic, the Se117 protein appeared to be a structural protein associated with nucleocapsids of occlusion-derived SeMNPV virions but not of budded virus.

Characteristics of the transactivator gene ie1 of Spodoptera exigua multiple nucleopolyhedrovirus.

The genomic position and nucleotide sequence of the immediate early gene ie1 of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) were determined. The SeMNPV ie1 gene had the potential to encode a protein of 714 amino acids with a predicted molecular mass of 82.0 kDa, representing the largest baculovirus IE1 known to date. The similarity of SeMNPV IE1 with IE1 proteins from other baculoviruses was restricted to the basic C-terminal two-thirds of the protein, which is involved in DNA binding. A single ie1 transcript of 2.5 kb was detected at 1 h p.i., peaking at 8 h p.i. and disappearing at 24 h p.i. From 8 h p.i. onwards a 1.7 kb transcript was detected, which became more abundant late in infection. Primer extension analysis revealed the use of several start sites early and late in infection. One of these was located in the promoter motif CAGT located at position -15 relative to the translational start codon, most intensively used at 8 h p.i. Four kb upstream of the SeMNPV ie1 gene, ORF xd244 was identified with homology to the used left ie0 exon of AcMNPV, OpMNPV and LdMNPV. Phylogenetic analysis indicated that the SeMNPV IE1 protein shared the most recent ancestor with its HzSNPV and LdMNPV homologues, although their IE1 proteins had diverged considerably from each other and from other baculoviruses.

A novel baculovirus envelope fusion protein with a proprotein convertase cleavage site.

The entry mechanism of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), a group II NPV, in cultured cells was examined. SeMNPV budded virus (BV) enters by endocytosis as do the BVs of the group I NPVs, Autographa californica (Ac) MNPV and Orgyia pseudotsugata (Op) MNPV. In group I NPVs, upon infection acidification of the endosome triggers fusion of the viral and endosomal membrane, which is mediated by the BV envelope glycoprotein GP64. However, the SeMNPV genome lacks a homolog of GP64 envelope fusion protein (EFP). A functional homolog of the OpMNPV GP64 EFP was identified in SeMNPV ORF8 (Se8; 76 kDa) and appeared to be the major BV envelope protein. Surprisingly, a 60-kDa cleavage product of this protein is present in the BV envelope. A furin-like proprotein convertase cleavage site (R-X-K/R-R) was identified immediately upstream of the N-terminus of the mature Se8 protein and this site was also conserved in the Lymantria dispar (Ld) MNPV homolog (Ld130) of Se8. Syncytium formation assays showed that Se8 and Ld130 alone were sufficient to mediate membrane fusion upon acidification of the medium. Furthermore, C-terminal GFP-fusion proteins of Se8 and Ld130 were primarily localized in the plasma membrane of insect cells. This is consistent with their fusogenic activity and supports the conclusion that the Se8 gene product is a functional homolog of the GP64 EFP.

Identification, sequence analysis and phylogeny of the lef-2 gene of Helicoverpa armigera single-nucleocapsid baculovirus.

The baculovirus late expression factor 2 (LEF-2) is involved in DNA replication, and most likely function as a primase processivity factor. Lef-2 genes have been found in multinucleocapsid nucleopolyhedroviruses (MNPVs) and in granuloviruses (GVs), but not yet in single-nucleocapsid NPV (SNPV). Here, a lef-2 gene homolog was identified from SNPV of Helicoverpa armigera (HearNPV). The open reading frame of the HearNPV lef-2 gene is 696 nucleotides long, encoding a putative protein of 232 amino acids with an M(r) of about 26 kDa. The 5'-noncoding region contains two early (CAGT) consensus motifs for transcription initiation and three TATA boxes. Lef-2 transcripts started at a C, 29 nucleotides upstream of a putative translational start. A putative polyA signal, AATAAA, was found 76 nucleotides downstream of the translation stop codon. The HearNPV lef-2 gene has a low but significant degree of amino acid sequence identity (30%) to the lef-2 genes of 15 other baculoviruses of which nine were newly determined. The N-terminal half of the LEF-2 proteins contains one (I) and the C-terminal half two (II and III) conserved domains. Sixteen amino acids are absolutely conserved in those LEF-2 investigated and are probably critical for LEF-2 function. A phylogenetic tree of 16 baculovirus LEF-2 proteins was constructed by using maximum parsimony analysis and appeared to be comparable to a tree for ecdysteroid UDP-glucosyl transferases (Chen et al., 1997a). The genomic location of the lef-2 genes relative to polyhedrin/granulin and the clade structure of the gene trees suggest that genome organization and gene phylogeny are useful parameters to study the evolutionary history of baculoviruses. These two independent approaches also give a more complete picture of the ancestral relationship among baculovirus.