RESUMO
Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2a, E, GP3, and GP4. Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion.
Assuntos
Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteínas do Envelope Viral/fisiologia , Montagem de Vírus , Animais , Linhagem Celular , Cricetinae , Imunoprecipitação , Glicoproteínas de Membrana/análise , Mutagênese Sítio-Dirigida , Nucleocapsídeo/química , Reação em Cadeia da Polimerase , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/crescimento & desenvolvimento , Ultracentrifugação , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
A set of neutralizing monoclonal antibodies (mAbs) directed against the GP(5) protein of European type porcine reproductive and respiratory syndrome virus (PRRSV) has been produced previously (Weiland et al., 1999). This set reacted with a plaque-purified virus (PPV) subpopulation of Dutch isolate Intervet-10 (I-10), but not with the European prototype PRRSV LV. In order to map the neutralization epitope in the GP(5) protein of the PPV strain, the ORF5 nucleotide sequence of PPV was determined. When the amino acid sequence derived from this nucleotide sequence was compared with that of PRRSV LV, four amino acid differences were found. Using site-directed mutagenesis, we showed that a proline residue at position 24 of the GP(5) sequence of the PPV strain enabled recognition by the neutralizing mAbs. Pepscan analysis demonstrated that the epitope recognized by the neutralizing mAbs stretched from residues 29 to 35. Surprisingly, the reactivity of the mAbs in the Pepscan system was independent of the presence of a proline in position 24. Moreover, residue 24 is located within the predicted signal peptide, implying that either the signal peptide is not cleaved or is cleaved due to the presence of Pro(24) such that the epitope remains intact. Our results demonstrate the presence of a neutralization epitope in the N-terminal ectodomain of the GP(5) protein of PRRSV and imply a role for the ectodomain of GP(5) in the infection of PRRSV.
Assuntos
Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Antígenos Virais/química , Antígenos Virais/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Cricetinae , DNA Viral/genética , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Técnicas In Vitro , Dados de Sequência Molecular , Mutação , Testes de Neutralização , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Prolina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sus scrofa , Transfecção , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
The aim of this study was to identify the receptor(s) for PRRSV on porcine alveolar macrophages (PAMs) by producing monoclonal antibodies (MAbs) against these cells. Hybridoma supernatants were selected for their ability to block PRRSV infection. Four MAbs, 1-8D2, 9.4C7, 9.9F2, and 3-3H2 inhibited infection and recognised cell surface, PAM-specific antigens as shown by immunofluorescence and immunoperoxidase monolayer assay. These MAbs were then used to identify cellular proteins involved in PRRSV infection by radioimmunoprecipitation assays (RIPAs). MAbs 1-8D2 and 9.9F2 each recognised a 150 kDa-polypeptide doublet, while MAbs 9.4C7 and 3-3H2 both recognised a 220 kDa-polypeptide. Glycosidase treatment demonstrated all these polypeptides to be N-glycosylated. Thus, multiple glycoproteins appear to be involved in infection of PAMs by PRRSV.
