RESUMEN
Genome-wide RNA interference (RNAi) screening is an emerging and powerful technique for genetic screens, which can be divided into arrayed RNAi screen and pooled RNAi screen/selection based on different screening strategies. To date, several genome-wide RNAi screens have been successfully performed to identify host factors essential for influenza virus replication. However, the host factors identified by different research groups are not always consistent. Taking influenza virus screens as an example, we found that a number of screening parameters may directly or indirectly influence the primary hits identified by the screens. This review highlights the differences among the published genome-wide screening approaches and offers recommendations for performing a good pooled shRNA screen/selection.
RESUMEN
SUMOylation is an important posttranslational modification for regulation of cellular functions and viral replication. Here, we report that protein SUMOylation regulates the replication of influenza A virus at the steps of viral maturation and assembly. Knocking down the SUMO-conjugating enzyme Ubc9 resulted in the reduction of virus production. Dissection of the virus life cycle revealed that SUMOylation is involved in the processes of virus maturation and assembly. The viral matrix protein M1 is SUMOylated at K242. A virus carrying the SUMO-defective M1 produced a lower titer of virus, while its viral proteins and viral RNA (vRNA) accumulated in the cells. Furthermore, the mechanistic studies showed that the SUMOylation of M1 is required for the interaction between M1 and viral RNP (vRNP) to form the M1-vRNP complex. The lack of M1 SUMOylation prevented the nuclear export of vRNP and subsequent viral morphogenesis. Taken together, our findings elucidate that the maturation and assembly of influenza A virus is controlled by the SUMO modification of M1 protein. Therefore, we suggest that M1 can serve as a target for developing a new generation of drugs for flu therapy.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus de la Influenza A/fisiología , Sumoilación , Proteínas de la Matriz Viral/metabolismo , Ensamble de Virus , Replicación Viral , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Perros , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Virus de la Influenza A/ultraestructura , Microscopía Electrónica de Transmisión , ARN Viral/genética , ARN Viral/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas Virales/metabolismoRESUMEN
Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by hepatitis C virus (HCV). However, the molecular mechanism of this synergy remains unclear. Here, we provide the first evidence that Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor formation by alcohol-induced endotoxemia. We also identify Nanog, the stem/progenitor cell marker, as a novel downstream gene up-regulated by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive LPS injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome. Taken together, our study demonstrates a TLR4-dependent mechanism of synergistic liver disease by HCV and alcohol and an obligatory role for Nanog, a TLR4 downstream gene, in HCV-induced liver oncogenesis enhanced by alcohol.