RESUMEN
Koala retrovirus (KoRV) is a gammaretrovirus that is currently endogenizing into koalas. Studies on KoRV infection have been hampered by the lack of a replication-competent molecular clone. In this study, we constructed an infectious molecular clone, termed plasmid pKoRV522, of a KoRV isolate (strain Aki) from a koala reared in a Japanese zoo. The virus KoRV522, derived from pKoRV522, grew efficiently in human embryonic kidney (HEK293T) cells, attaining 10(6) focus-forming units/ml. Several mutations in the Gag (L domain) and Env regions reported to be involved in reduction in viral infection/production in vitro are found in pKoRV522, yet KoRV522 replicated well, suggesting that any effects of these mutations are limited. Indeed, a reporter virus pseudotyped with pKoRV522 Env was found to infect human, feline, and mink cell lines efficiently. Analyses of KoRV L-domain mutants showed that an additional PPXY sequence, PPPY, in Gag plays a critical role in KoRV budding. Altogether, our results demonstrate the construction and characterization of the first infectious molecular clone of KoRV. The infectious clone reported here will be useful for elucidating the mechanism of endogenization of the virus in koalas and screening for antiretroviral drugs for KoRV-infected koalas.
Asunto(s)
Clonación Molecular , Gammaretrovirus/genética , Gammaretrovirus/aislamiento & purificación , Phascolarctidae/virología , Infecciones por Retroviridae/veterinaria , Secuencia de Aminoácidos , Animales , Gatos , Línea Celular , Gammaretrovirus/fisiología , Células HEK293 , Humanos , Japón , Visón , Datos de Secuencia Molecular , Infecciones por Retroviridae/virología , Alineación de Secuencia , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Liberación del Virus , Replicación ViralRESUMEN
RD-114 virus is a replication-competent feline endogenous retrovirus (ERV). RD-114 virus had been thought to be xenotropic; however, recent findings indicate that RD-114 virus is polytropic and can infect and grow efficiently in feline cells. Receptor(s) for RD-114 virus has not been identified and characterized in cats. In this study, we confirmed that two feline sodium-dependent neutral amino acid transporters (ASCTs), fASCT1 and fASCT2, function as RD-114 virus receptors. By chimeric analyses of feline and murine ASCTs, we revealed that extracellular loop 2 of both fASCT1 and fASCT2 determines the susceptibility to RD-114 virus. Further, we revealed ubiquitous expression of these genes, consistent with the general metabolic role of the ASCT molecules. Our study indicates that RD-114 virus may reinfect tissues and cells in cats, once the virus is activated. Implications of the involvement of RD-114 virus in feline oncogenesis are also discussed.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/metabolismo , Retrovirus Endógenos/patogenicidad , Receptores Virales/metabolismo , Infecciones por Retroviridae/veterinaria , Sistema de Transporte de Aminoácidos ASC/genética , Animales , Enfermedades de los Gatos/virología , Gatos/virología , Línea Celular , Retrovirus Endógenos/metabolismo , Retrovirus Endógenos/fisiología , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Receptores Virales/genética , Infecciones por Retroviridae/virología , Análisis de Secuencia de ADN , Activación ViralRESUMEN
Koala retrovirus (KoRV) is a unique gammaretrovirus that is currently endogenizing into its host and considered to be associated with leukemia, lymphoma and immunosuppression in koalas (Phascolactos cinereus). In this study, it was demonstrated that WWP2 or WWP2-like E3 ubiquitin ligases possessing the WW domain closely related to WWP2 and Vps4A/B are involved in KoRV budding. These data suggest that KoRV Gag recruits the cellular endosomal sorting complex required for transport machinery through interaction of the PPPY L-domain with the WW domain(s) of WWP2 and that progeny virions are released from cells by utilizing the multivesicular body sorting pathway.