RESUMO
An Autographa californica nuclear polyhedrosis virus gene encoding an occlusion-derived virus (ODV) envelope protein of 56 kDa was identified and sequenced. Transcription initiates from a conserved baculovirus late motif (ATAAG) with transcripts detected from 16 through 72 hr p.i. The protein is detected in infected cell extracts from 36 hr p.i. Western blot assay of ODV, BV, viral envelope, and nucleocapsid preparations coupled with immunoelectron microscopy reveal that this protein localizes to the ODV envelope. This protein is named ODV-E56 to identify its viral origin, envelope location, and apparent molecular weight. ODV-E56 is enriched in viral induced intranuclear microvesicles as determined by immunogold labeling. A mutant was constructed with the C-terminal portion of the protein replaced with beta-galactosidase. The fusion protein, E56-beta-gal, locates to the viral nucleocapsids and not to the ODV envelope or intranuclear microvesicles. This suggests that the signals necessary for transport and/or retention into these structures lies within the C-terminal portion of ODV-E56. Additionally, both ODV-E56 and E56-beta-gal are enriched in electron dense regions that cluster around the inner nuclear membrane and within the nucleoplasm.
Assuntos
Proteínas de Membrana , Nucleopoliedrovírus/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Viral , Genes Virais , Glicosilação , Corpos de Inclusão Viral/genética , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Nucleopoliedrovírus/química , Nucleopoliedrovírus/ultraestrutura , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/ultraestrutura , Spodoptera , Proteínas do Envelope Viral/análise , beta-Galactosidase/genéticaRESUMO
A portion of equine herpesvirus type 1 (EHV-1) gene 28, which is homologous to herpes simplex virus type 1 gene UL32, was expressed using a prokaryotic system to yield a fusion protein which reacted on Western blots with P19, a monoclonal antibody (MAb) that reacts with EHV-1 glycoprotein 300 (gp300), confirming that this gene encodes gp300. Hydrophobicity analysis showed that gp300 is a glycoprotein with multiple hydrophobic domains that might interact with, or span, the membrane several times. As such, it may represent the first member of a new family of herpesvirus glycoproteins to be identified as a virus structural component. Gp300 was also shown to be modified by palmitic acid residues, and a second MAb (1G12) directed against gp300 inhibited fusion between EHV-1-infected cells.
Assuntos
Genes Virais/genética , Herpesvirus Equídeo 1/genética , Proteínas do Envelope Viral/genética , Acilação , Sequência de Aminoácidos , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/farmacologia , Sequência de Bases , Fusão Celular/efeitos dos fármacos , Escherichia coli/genética , Dados de Sequência Molecular , Plasmídeos/genética , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Virais de Fusão/genética , Proteínas Virais/genéticaRESUMO
The DNA sequence of the equine herpesvirus type 1 (EHV-1) gD gene homologue has been determined for the strain Ab1 and compared with previously published sequences. A portion of the gene has been located to a region of the genome which also encodes homologues of the herpes simplex virus type 1 genes for gE and gI and is known to encode an epitope of the virion protein gp17/18. Analysis of the EHV-1 strain Kentucky A (KyA) by DNA hybridization showed the presence of a gD gene homologue and established the absence of genes for gI and gE. Western blot analysis, however, showed that KyA virus particles contain gp17/18, thus indicating that this protein is encoded by the gD gene homologue. The KyA gp17/18 was found to be smaller than that detected in other strains and this is accounted for by a frameshift mutation in the KyA sequence relative to Ab1. The mutation in the KyA strain results in an altered C-terminal sequence and could explain the apparent structural differences suggested by the reactivities with monoclonal antibodies (MAbs). We have also expressed part of the Ab1 gD gene as a fusion protein with glutathione S-transferase in Escherichia coli and shown that this reacts with the MAb 5H6 originally used to map gp17/18. These experiments establish that gp17/18 is encoded by the gD gene homologue.
Assuntos
Herpesvirus Equídeo 1/genética , Simplexvirus/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Dados de Sequência Molecular , Família Multigênica/genética , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência do Ácido NucleicoRESUMO
Monoclonal antibodies (MAbs) specific for equine herpesvirus type 1 (EHV-1) glycoprotein 60 (gp60) and gp 17/18 (F3132 and 5H6 respectively) were found to react with the same protein, which was identified as a homologue of herpes simplex virus type 1 gD. MAb F3132 strongly neutralized virus infectivity and inhibited the penetration of the virus into the cell. The effects on penetration were shared with three other MAbs against this protein (P68, F3116 and F3129), but no effect on virus penetration was found with any other anti-EHV-1 MAb tested. The level of glycosylation of gp60 was analysed using glycanase enzymes and glycosylation inhibitors, and consisted of mainly N-linked carbohydrate. The M(r) of non-N-glycosylated gp60 was 50K.
Assuntos
Herpesvirus Equídeo 1/genética , Simplexvirus/genética , Proteínas do Envelope Viral/genética , Replicação Viral/genética , Animais , Anticorpos Monoclonais , Carboidratos/análise , Glicosilação , Infecções por Herpesviridae/metabolismo , Herpesvirus Equídeo 1/patogenicidade , Dados de Sequência Molecular , Testes de Neutralização , Homologia de Sequência do Ácido Nucleico , Proteínas do Envelope Viral/imunologia , VirulênciaRESUMO
The DNA fragments representing the entire short unique region and part of the repeat sequences of the equine herpesvirus type-1 genome were cloned into plasmid vectors. The approximate positions of the junctions between the short unique region and the inverted repeats were then located by restriction endonuclease mapping. Two open reading frames coding for potential glycoproteins have been identified within the short unique region, using DNA sequence analysis. The predicted amino acid sequences of these open reading frames had extensive homology to the herpes simplex virus glycoproteins gE and gI and the related glycoproteins of pseudorabies virus and varicella-zoster virus.