RESUMO
Attempts were made to linearize the DNA of Choristoneura fumiferana (Cf) multicapsid nucleopolyhedrovirus (MNPV), in order to improve the efficiency of generation of recombinant viruses after transfection. A unique site for the restriction enzyme Sse83871 was found in ORF p48. The requirement for this ORF during virus replication was investigated by molecular analyses including sequencing, transcriptional analysis and inactivation by insertion of marker genes. Sequence analysis showed that ORF p48 consists of 1233 nucleotides encoding a potential protein of 47.88 kDa. The proteins encoded by ORF p48 from CfMNPV and Orgyia pseudotsugata MNPV contain 411 amino acids while that from CfDEFNPV (a virus that is defective for infection by the per os route) is slightly smaller, at 408 amino acids. Transcriptional and primer extension analyses showed that the mRNA is initiated from a typical baculovirus late gene ATAAG motif. The mRNA was detected at 24 h post-infection (p.i.), reached maximum levels at 48 h p.i. and declined by 96 h p.i., which confirmed the late property of the gene. Inactivation of the gene was attempted by inserting a cassette containing either the gene encoding beta-galactosidase or that encoding green fluorescent protein. Blue or fluorescent green plaques of infected cells were observed after transfection. Attempts to generate a plaque-purified virus were not successful. Restriction enzyme analysis showed that the marker genes were inserted randomly at positions other than the p48 locus. This indicated that the gene may be needed for virus replication. The gene is relatively well conserved among baculoviruses but its function remains unclear.
Assuntos
Genes Virais , Genoma Viral , Lepidópteros/virologia , Nucleopoliedrovírus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Recombinante/análise , DNA Recombinante/genética , DNA Viral/análise , DNA Viral/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Alinhamento de SequênciaRESUMO
A gene encoding a capsid-associated viral structural protein has been identified and sequenced in the genome of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV). The gene has a 1872 nucleotide open reading frame (ORF) encoding 624 amino acids with a predicted molecular mass of 71.4 kDa. Transcription, which appeared to be initiated from a conserved GTAAG motif of baculovirus late genes, was detected at 12 h, reached a maximum at 48 h and declined at 72 h post-infection (p.i.). Part of the ORF was cloned in frame into a prokaryotic expression vector, pMAL-c2, and the fusion protein was used to generate antibodies in rabbits. It was shown, with the aid of the polyclonal antiserum, that this viral protein was detectable at 24 h p.i. in infected cells. The protein appeared as an 82 kDa band in occlusion-derived virus and as an 82 kDa band and a 72 kDa band in budded virus. Amino acid sequence comparisons revealed that this ORF had high homology with the ORF p87 (77% similarity) of Orgyia pseudotsugata (Op) MNPV and the ORF p80 (60% similarity) of Autographa californica (Ac) MNPV. Immunoblots confirmed that the CfMNPV protein had antigenic similarities to the P87 protein of OpMNPV, but not to the P80 of AcMNPV.
Assuntos
Proteínas do Capsídeo , Capsídeo/genética , Nucleopoliedrovírus/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Sequência de Bases , Western Blotting , Genes Virais , Dados de Sequência Molecular , Peso Molecular , Mariposas/virologia , Fases de Leitura Aberta , RNA Mensageiro/genética , RNA Viral/genética , Mapeamento por Restrição , Alinhamento de Sequência , Solubilidade , Transcrição GênicaRESUMO
We have identified the lef-1 genes from two multiple nucleopolyhedroviruses that infect natural populations of Choristoneura fumiferana. The lef-1 genes in both viruses are directly upstream and in the opposite orientation of their respective ecdysteroid UDP-glucosyltransferase (egt) genes. This gene organization pattern is similar to that found in the genomes of AcMNPV and of OpMNPV. As well, the coding regions and putative protein sequences share a high degree of similarity. Alignment of the predicted amino acid sequences of all known baculovirus lef-1 genes suggests that the LEF-1 proteins have a relatively high degree of conservation, particularly at four identified and distinct domains. Moreover, LEF-1 proteins bear clear similarity to some eukaryotic primases, predominately at three of the four domains where certain amino acids are absolutely conserved.