This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.This project aims to directly tag viral proteins with GFP in order to follow them in the infected host and find their interacting partners. In this respect, the methodology developed for the visualization and isolation of protein complexes (described on project #177) was applied for the study of Sindbis alphavirus replication complex in rat fibroblasts. Sindbis is a single strand alphavirus, arthropod borne, usually having birds as their primary vertebrate hosts and therefore, with a wide geographic distribution. The structure of the virus contains a nonstructural polyprotein, formed of 4 nonstructural proteins (nsP1, nsP2, nsP3, and nsP4), and a structural domain. The formation of the virus replication complex involves the breakage of the nsP3/nsP4 bond, synthesis of the minus strand RNA, cleavage of the nsP1/nsP2 and nsP2/nsP3 bonds, and conformational changes. Our immediate interest is the identification of the host proteins involved in the formation of this replication complex.Our first results from pullouts of the nsP3-GFP complex show that this method enabled the isolation of the nonstructural viral proteins from whole cell lysate together with other proteins of potential interest.
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