Sequencing and Characterisation of p74 Gene in Two Isolates of Anticarsia Gemmatalis MNPV

P74 is a protein encoded in the genome of baculoviruses, associated with the envelopes of occluded virus. Its presence proved to be essential for per os infection. In first place, in this work we designed two universal primers to amplify a sequence region of the p74 ORF in baculoviruses from different classification groups. Then, by the use of these amplicons we obtained the complete sequence of the p74 locus from two isolates of AgMNPV, 2D (Brazil) and SF (Argentina). In the flanking regions we determined the complete sequence of p10 gene and a portion of p26 gene. Comparing both p74 sequence data (ORFs of 1935 bp) we found fifteen nucleotide changes that result in six amino acid changes. Comparisons of AgMNPV p74s with other baculovirus homologous genes indicate a close relationship with other group I Nucleopolyhedrovirus, in particular CfDEFNPV. These results were based on ORF sequence, amino acid sequence and gene order. The predictive studies about secondary structure and hydrophobic index point at six regions potentially associated to its function or native conformation. Finally, the detection of p74 mRNA after virus DNA replication confirms a late expression pattern.     

Sequence Analysis of a 5.1 kbp Region of the Spodoptera frugiperda Multicapsid Nucleopolyhedrovirus Genome that Comprises a Functional Ecdysteroid UDP-Glucosyltransferase (egt) Gene

The ecdysteroid UDP-glucosyltransferase gene from the Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) was identified using degenerate primers whose sequence were derived from conserved regions of the EGT proteins encoded by other baculoviruses. Analysis of the gene sequence revealed the presence of an open reading frame (ORF) with potential to encode a polypeptide of 525 amino acids. Promoter sequences typical of baculovirus genes were found in the 5 region of this ORF. A polyadenylation signal was identified downstream the translation stop codon. A transient expression assay showed that the product of this ORF was able to conjugate glucose from UDP-glucose with ecdysone confirming that the gene identified was indeed the SfMNPV egt gene. The SfMNPV egt gene and the sequences of other baculovirus egt genes were used to infer a phylogenetic tree. The nucleotide sequence of the entire BamHI fragment that contains the SfMNPV egt gene was determined. Search of the available sequence databases suggested that, besides the egt gene, this region contains 5 ORFs similar to the baculovirus genes gp37 (fusolin), to ptp2 and to ORFs 28, 29, and 30 of Spodoptera exigua multicapsid nucleopolyhedrovirus. Both the phylogenetic analysis of the egt genes and the gene order of the region that flanks the egt gene indicated that SfMNPV is closely related to the baculoviruses that infects S. exigua and Mamestra configurata.     

Sequence and organization of the Mamestra configurata nucleopolyhedrovirus genome

The nucleotide sequence of the genome of the nucleopolyhedrovirus (NPV) from Mamestra configurata (MacoNPV, isolate 90/2), a group II NPV, was determined and analyzed. The MacoNPV DNA genome consists of 155,060 bp and has an overall G+C content of 41.7%. Computer-assisted analysis predicted 169 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. BLAST searches and comparisons with completely sequenced baculoviruses indicated that there were 66 ORFs conserved among the nine baculoviruses compared and an additional 17 ORFs were conserved among the NPVs. The gene content and gene arrangement in MacoNPV were most similar to those of SeMNPV, including two putative odv-e66 and p26 gene homologues. However, in contrast to SeMNPV, 8 ORFs with homology to baculovirus repeat ORFs (bro) and single copies of enhancin and conotoxin-like protein ORFs were found in MacoNPV. The MacoNPV genome contained four homologous regions, each with 10 to 17 repeated sequences. Each repeat was 60 to 86 nucleotides in length and contained an approximately 43-bp-long imperfect palindrome. There were 13 ORFs unique to MacoNPV, ranging from a small ORF of 196 bp to larger ORFs of up to 1047 bp, and many of these contained typical early and late baculovirus consensus promoters.     

Nucleotide sequence and phylogenetic analyses of the DNA polymerase gene of Anticarsia gemmatalis nucleopolyhedrovirus

The DNA polymerase from Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) was identified and sequenced, and its amino acid sequence was compared with other viral DNA polymerases to identify conserved regions and to reconstruct a phylogenetic tree. The sequence analysis of the AgMNPV DNA polymerase gene revealed the presence of a 2976 nucleotides open reading frame (ORF) encoding a polypeptide of 991 amino acid residues with a predicted molecular mass of 114.7 kDa. Among the baculovirus DNA polymerase genes identified to date, the AgMNPV DNA polymerase gene shared maximum amino acid sequence identity with the DNA polymerase gene of Choristoneura fumiferana nucleopolyhedrovirus defective strain (CfDEFNPV) (94%). The alignment of 140 virus sequences, 23 of them from baculovirus, showed that, of the 10 conserved regions identified, 5 are exclusive to baculoviruses (R1, R5, R9, R6 and R10), only 2 of them (R6 and R10) previously described as such in the literature. Our analysis, based on their positions in the AgMNPV DNA polymerase model, suggests that R9 and R10 could interact with DNA. Phylogenetic analysis of DNA polymerase sequences places the enzyme from AgMNPV within the cluster containing the polymerases of Group I Nucleopolyhedrovirus and suggests that the AgMNPV DNA polymerase is more closely related to that of CfDEFNPV than to those of other baculoviruses.     

Sequence analysis of the complete genome of Trichoplusia ni single nucleopolyhedrovirus and the identification of a baculoviral photolyase gene

The genome of the Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), a group II NPV which infects the cabbage looper (T. ni), has been completely sequenced and analyzed. The TnSNPV DNA genome consists of 134,394 bp and has an overall G + C content of 39%. Gene analysis predicted 144 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. Comparisons with previously sequenced baculoviruses indicate that 119 TnSNPV ORFs were homologues of previously reported viral gene sequences. Ninety-four TnSNPV ORFs returned an Autographa californica multiple NPV (AcMNPV) homologue while 25 ORFs returned poor or no sequence matches with the current databases. A putative photolyase gene was also identified that had highest amino acid identity to the photolyase genes of Chrysodeixis chalcites NPV (ChchNPV) (47%) and Danio rerio (zebrafish) (40%). In addition unlike all other baculoviruses no obvious homologous repeat (hr) sequences were identified. Comparison of the TnSNPV and AcMNPV genomes provides a unique opportunity to examine two baculoviruses that are highly virulent for a common insect host (T. ni) yet belong to diverse baculovirus taxonomic groups and possess distinct biological features. In vitro fusion assays demonstrated that the TnSNPV F protein induces membrane fusion and syncytia formation and were compared to syncytia formed by AcMNPV GP64.     

Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome

The complete Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) genome contained 139,342 bp with a G+C content of 42.7%, and 141 putative open reading frames (ORFs) or genes of 150 nucleotides or greater that showed minimal overlap. Ninety-six ORFs had homologues in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), 16 had homologues in other baculoviruses, and 29 were unique to SpltMNPV. The homologues of ubiquitin and gp37 are fused in SpltMNPV. The genome lacked a homologue of the major budded virus glycoprotein gene gp64, but it contained a homologue of ORF130 of Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV). There were two homologues of AcMNPV ORF2 (bro gene), and a DnaJ protein gene (SpltORF39) in which the N-terminus showed homologies with the J domain of DnaJ family proteins. Seventeen homologous regions (hrs) were identified, each containing 2-29 palindromic repeats, with an average length of 534 bp and base content (G+C%) of 33.0.     

Molecular characterization of genes in the GP41 region of baculoviruses and phylogenetic analysis based upon GP41 and polyhedrin genes.

A newly sequenced Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) gp41 gene was used to reconstruct the phylogeny for gp41 by comparison with Autographa californica MNPV, Bombyx mori MNPV, Helicoverpa zea single nucleopolyhedrovirus (SNPV), Lymantria dispar MNPV, Orgyia pseudotsugata MNPV and Spodoptera frugiperda MNPV. The 3.5 kb fragment of the AgMNPV gp41 region not only contained the gp41 gene but also three other open reading frames that had significant homology with the very late factor (vlf-1) of baculoviruses, AcMNPV ORF78, AcMNPV ORF79, and one partial open reading frame homologous to AcMNPV ORF81. The reconstructed phylogenetic tree of baculovirus gp41 genes compared with the polyhedrin gene tree produced similar topologies. Two other phylogenetic trees were reconstructed based on either combined gp41 and polyhedrin nucleotide sequences (total evidence) or combined evolutionary histories of both genes (strict consensus tree). The former had an identical tree topology as the gp41 gene tree alone, and the latter lost resolution in the branch of AcMNPV and BmMNPV. Mutation rate analysis showed the gp41 gene had a higher nucleotide substitution rate than the polyhedrin gene, implying that the polyhedrin gene may have a different selection constraint than the gp41 gene. Both genes have nonsynonymous/synonymous substitution values close to 0.1, similar to other DNA viruses.     

Identification of a Gene Cluster within the Genome of Chilo Iridescent Virus Encoding Enzymes Involved in Viral DNA Replication and Processing

The nucleotide sequence of the genome of Chilo iridescent virus (CIV) between the genome coordinates 0.974 and 0.101 comprising 27,079 bp was determined. Computer-assisted analysis of the DNA sequence of this particular region of the CIV genome revealed the presence of 42 potential open reading frames (ORFs) with coding capacities for polypeptides ranging from 50 to 1,273 amino acid residues. The analysis of the amino acid sequences deduced from the individual ORFs resulted in the identification of 10 potential viral genes that show significant homology to functionally characterized proteins of other species. A cluster of five viral genes that encode enzymes involved in the viral DNA replication was identified including the DNA topoisomerase II (A039L, 1,132 amino acids (aa)), the DNA polymerase (ORF A031L, 1,273 aa), a helicase (ORF A027L, 530 aa), a nucleoside triphosphatase I (ORF A025L, 1,171 aa), and an exonuclease II (ORF A019L, 624 aa), all ORFs possessing the same genomic orientation. The DNA polymerase of CIV showed the highest homology (24.8% identity) to the DNA polymerase of lymphocystis disease virus lymphocystis disease virus 1 (LCDV-1), a member of the family Iridoviridae, indicating the close relatedness of the two viruses. In addition, four putative gene products were found to be significantly homologous to previously identified hypothetical proteins of CIV.     

Complete nucleotide sequence of rose yellow leaf virus,a new member of the family Tombusviridae

The genome of the rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides long and to contain seven open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to encode an 87-kDa (p87) protein that has amino acid similarity to the RNA-dependent RNA polymerase (RdRp) of members of the family Tombusviridae. ORFs 3 and 4 have no significant amino acid similarity to known functional viral ORFs. ORF5 encodes a 6-kDa (p6) protein that has similarity to movement proteins of members of the Tombusviridae. ORF5A has no conventional start codon and overlaps with p6. A putative +1 frameshift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein that has high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CP) of members of the Tombusviridae. ORF7 has no significant amino acid similarity to known viral ORFs. Phylogenetic analysis of the RdRp amino acid sequences grouped RYLV together with the unclassified Rosa rugosa leaf distortion virus (RrLDV), pelargonium line pattern virus (PLPV), and pelargonium chlorotic ring pattern virus (PCRPV) in a distinct subgroup of the family Tombusviridae.     

The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes.

A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.