RESUMO
We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the -492 and -357 versions contains sequence motifs important for the level of the lacZ reporter gene expression.
Assuntos
Proteínas Imediatamente Precoces/genética , Nucleopoliedrovírus/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Fusão Gênica Artificial , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Sequência Conservada/genética , DNA Viral/química , DNA Viral/genética , Escherichia coli/genética , Genes Reporter , Proteínas Imediatamente Precoces/fisiologia , Dados de Sequência Molecular , Mariposas/virologia , Nucleopoliedrovírus/fisiologia , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Ativação Transcricional/fisiologia , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
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.
Assuntos
Genes Virais , Nucleopoliedrovírus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Viral/genética , Genoma Viral , Lepidópteros/virologia , Dados de Sequência Molecular , Nucleopoliedrovírus/classificação , Nucleopoliedrovírus/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase , Transcrição Gênica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
The gp64 locus of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate Santa Fe (AgMNPV-SF) was characterised molecularly in our laboratory. To this end, we have located and cloned a AgMNPV-SF genomic DNA fragment containing the gp64 gene and sequenced the complete gp64 locus. Nucleotide sequence analysis indicated that the AgMNPV gp64 gene consists of a 1500 nucleotide open reading frame (ORF), encoding a protein of 499 amino acids. Of the seven gp64 homologues identified to date, the AgMNPV gp64 ORF shared most sequence similarity with the gp64 gene of Orgyia pseudotsugata MNPV. The GP64 from AgMNPV is the smallest baculoviral envelope glycoprotein found to date, differing in 10 or more residues from the other group I nucleopolyhedroviruses. The biological activity of AgMNPV GP64 protein was assessed by cell fusion assays in UFL-AG-286 cells using the obtained recombinant plasmids. In the upstream and downstream regions, relative to the gp64 ORF, we found different conserved transcriptional and post-transcriptional regulatory elements, respectively.
