An RNAi screen for host factors involved in actin-dependent spread of vaccinia virus

DIEGO E. ALVAREZ; HERVE AGAISSE

Cozumel

Encuentro; PEW Programs in the Biomedical Sciences 2011 Annual Meeting; 2011

Vaccinia virus is the prototype of poxvirus family and was successfully employed as a vaccine to eradicate smallpox. Poxviruses are large, enveloped, double-stranded DNA virus. Virus morphogenesis begins in the cell cytoplasm at viral factories to produce intrcellular mature virions (IMV) that are subsequently transported to the trans-Golgi network and early endosome to acquire a two membrane wrapping yielding intracellular enveloped virions (IEV). After transport on microtubules, IEV fuse with the plasma membrane releasing extracellular virions (EV). The cell-associated EV is capable of initiating an outside-in signaling cascade that promotes actin polymerization beneath the plasma membrane, facilitating cell-to-cell spread of the virus. While several players involved in virus induced actin polymerization have been identified, the molecular mechanism that triggers this process is poorly understood. In order to identify novel host factors required for actin-based spread of vaccinia virus we developed an approach relying on the siRNA methodology and high-throughput imaging of viral infection. To this end we designed a recombinant vaccinia virus that expresses a fluorescently tagged core protein (mCherry-A5L). This virus allows us to easily monitor spreading in epithelial cells. In order to validate our assay, we transfected cells with siRNAs targeting different host genes known to be involved in vaccinia virus induced actin polymerization. As expected, depletion of N-WASP or components of the Arp2/3 actin-nucleating complex impaired virus spread as compared to mock transfected cells. In order to distinguish genes involved in actin-dependent spread from those required in previous steps during viral replication, we developed a secondary assay to discriminate cell-associated EV at the plasma membrane and viral forms inside the infected cell. We introduced a triple FLAG epitope in the outer envelope portion of the EV membrane protein B5R of the virus. Cell-associated EV can be detected by immunostaining of non-permeabilized cells with an anti-FLAG antibody, while intracellular forms of the virus are not stained. Thus, candidate genes involved in actin-based spread must display a non-spreading phenotype and positive FLAG staining. We are currently using this assay to screen a siRNA library targeting the human kinases. The systematic identification of host factors involved in virus spread will provide new insights into how vaccinia virus exploits the cellular architecture to mobilize from a primary infected cell to the neighboring cells.