Structural and Cell-associated Proteins of Lassa Virus

Abstract

Summary Lassa virus was purified from culture fluids of infected CV-1 monkey kidney cells and its structural proteins analysed by polyacrylamide gel electrophoresis. Stained gels showed a typical arenavirus profile, with a prominent protein of molecular weight 60000, corresponding to the nucleocapsid protein N, and two faint broad bands with molecular weights of 45000 and 38000, the envelope glycoproteins G1 and G2. G1 and G2 were shown to be glycosylated by their ability to bind concanavalin A to nitrocellulose transfers of the separated proteins (‘Western blots’). N and G2 bound antibody from guinea-pig or human convalescent sera but G1 was inactive, presumably as a result of denaturation. This technique also revealed other apparently virus-specific minor bands with molecular weights of 76000 and 68000. When Western blots of proteins of infected cells which had been lysed in SDS were probed with anti-Lassa virus serum or stained for glycoproteins, four virus-specific bands were apparent: the N, G1 and G2 proteins seen in purified virus, and a glycoprotein of molecular weight 72000 which probably corresponds to the envelope protein precursor (GPC) seen in other arenavirus systems. Immunoprecipitates from infected CV-1 cells labelled with [35S]methionine contained three major virus-specific proteins: the nucleocapsid protein N and proteins of 36000 and 24000 molecular weight (designated fN1 and fN2). Similar immunoprecipitates from Vero cells contained fN1 and fN2 and only very low levels of N. The polypeptides fN1 and fN2 are most probably fragments of N, since Western blots probed with anti-Lassa virus serum showed that lysis of cells in non-ionic detergent rather than SDS results in the appearance of fN2 with concomitant reduction or disappearance of N. These fragments do not exist in the intact cell, but are found as a consequence of rather specific proteolysis upon disruption under non-denaturing conditions. The proteolytic activity responsible was refractory to inhibition by phenylmethylsulphonyl fluoride, aprotinin, pepstatin A or sodium bisulphite, and was more active in Vero than in CV-1 cells.