Purification of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) and analyses of the structural proteins

Abstract

The Kaposis's sarcoma-associated herpesvirus (KSHV) infected BCBL-1 cell line, adapted to and grown in medium containing 10% horse serum, was induced to lytic replication with 12- O-tetradecanoylphorbol-13-acetate (TPA) for virus production. Supernatants from induced cells were filtered through a 0.45- μm filter and virions were concentrated by polyethylene glycol extraction and high speed centrifugation. The virus was purified by a glycerol gradient zonal centrifugation step followed by isopycnic separation using positive density–negative viscosity gradients. Two visible bands were detected after the final centrifugation step: an upper band that contained a homogenous population of purified virions and a lower band that contained aggregates of purified virus and other cellular debris. Fractionation of purified virion preparations by SDS-PAGE revealed 32 bands with estimated molecular weights between 19 and 280 K in silver stained gels. The glycoprotein bands in purified virus were identified with biotinylated lectins and horseradish peroxidase-labeled streptavidin. Two lectins were used to identify the KSHV glycoproteins: concanavalin A and Ricinus communis agglutinin I. Eight distinct glycoproteins were detected with these lectins. In addition, antisera from KS patients were used to detect immunoreactive proteins in purified virions. An apparent immunodominant band of M r 94 000 (94 K) was recognized by patients' antisera. Other proteins detected with some of the KS antisera tested corresponded to molecular weights of 57 K, 70 K, 180 K, 200 K and 240 K. The 94 K band was identified as gp94 by Endo F digestion.