RESUMO
Pestivirus envelope protein E2 is crucial to virus infection and accomplishes virus-receptor interaction during entry. However, mapping of E2 residues mediating these interactions has remained unexplored. In this study, to investigate the structure-function relationship for a ß-hairpin motif exposed to the solvent in the crystal structure of bovine viral diarrhea virus (BVDV) E2, we designed two amino acidic substitutions that result in a change of electrostatic potential. First, using wild type and mutant E2 expressed as soluble recombinant proteins, we found that the mutant protein had reduced binding to susceptible cells compared to wild type and diminished ability to inhibit BVDV infection, suggesting a lower affinity for BVDV receptors. We then analyzed the effect of ß-hairpin mutations in the context of recombinant viral particles. Mutant viruses recovered from cell culture supernatant after transfection of recombinant RNA had almost completely inhibited ability to re-infect susceptible cells, indicating an impact of mutations on BVDV infectivity. Finally, sequential passaging of the mutant virus resulted in the selection of a viral population in which ß-hairpin mutations reverted to the wild type sequence to restore infectivity. Taken together, our results show that this conserved region of the E2 protein is critical for the interaction with host cell receptors.
Assuntos
Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Substituição de Aminoácidos , Animais , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina/química , Sequências Repetidas Invertidas/fisiologia , Ligação Proteica , Proteínas do Envelope Viral/genéticaRESUMO
The BVDV glycoproteins gp48 and gp53 were expressed in the baculovirus eukaryotic system. Both recombinant proteins were recognized in western blot analysis by monoclonal antibodies and polyclonal serum. Immunofluorescent test demonstrated that gp53 was localized on the cell surface whereas gp48 was in the cytoplasm. The expressed proteins were extracted by non-denaturing detergent treatment. Rabbit antiserum raised against gp53 recombinant protein efficiently neutralized the virus. These results demonstrate that the recombinant proteins have immunological properties similar to those of the native viral protein and that they can be useful as diagnostic reagents.
Assuntos
Vírus da Diarreia Viral Bovina/química , Proteínas do Envelope Viral/isolamento & purificação , Animais , Western Blotting , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , Vetores Genéticos/genética , Soros Imunes , Rim/citologia , Masculino , Nucleopoliedrovírus/genética , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Spodoptera/citologia , Testículo/citologia , Transfecção , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
The BVDV glycoproteins gp48 and gp53 were expressed in the baculovirus eukaryotic system. Both recombinant proteins were recognized in western blot analysis by monoclonal antibodies and polyclonal serum. Immunofluorescent test demonstrated that gp53 was localized on the cell surface whereas gp48 was in the cytoplasm. The expressed proteins were extracted by non-denaturing detergent treatment. Rabbit antiserum raised against gp53 recombinant protein efficiently neutralized the virus. These results demonstrate that the recombinant proteins have immunological properties similar to those of the native viral protein and that they can be useful as diagnostic reagents.
Assuntos
Animais , Bovinos , Masculino , Coelhos , Proteínas do Envelope Viral/isolamento & purificação , Vírus da Diarreia Viral Bovina/química , Western Blotting , Linhagem Celular , Soros Imunes , Rim , Nucleopoliedrovírus , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Spodoptera , Testículo/citologia , Transfecção , Vetores Genéticos/genética , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologiaRESUMO
The BVDV glycoproteins gp48 and gp53 were expressed in the baculovirus eukaryotic system. Both recombinant proteins were recognized in western blot analysis by monoclonal antibodies and polyclonal serum. Immunofluorescent test demonstrated that gp53 was localized on the cell surface whereas gp48 was in the cytoplasm. The expressed proteins were extracted by non-denaturing detergent treatment. Rabbit antiserum raised against gp53 recombinant protein efficiently neutralized the virus. These results demonstrate that the recombinant proteins have immunological properties similar to those of the native viral protein and that they can be useful as diagnostic reagents.(AU)