CYTOSKELETAL PROTEINS INSIDE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 VIRIONS

We have identified three types of cytoskeletal proteins inside human i mmunodeficiency virus type 1 (HTV-1) virions by analyzing subtilisin-d igested particles. HIV-1 virions were digested with protease, and the treated particles mere isolated by sucrose density centrifugation. Thi s method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the pro teins inside the virion are protected from digestion by the viral lipi d envelope, they can be isolated and analyzed after treatment. Experim ents presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digeste d HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were ana lyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal pro teins are present in virions at different concentrations relative to t he molar level of Gag: actin (approximately 10 to 15%), ezrin and moes in (approximately 2%), and cofilin (approximately 2 to 10%). Our analy sis of proteins within virus particles detected proteolytic fragments of cw-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these proc essed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a fe w specific cytoskeletal proteins into virions, suggests an active inte raction between cytoskeletal and, viral proteins.