Human parainfluenza virus 3: purification and characterization of subviral components, viral proteins and viral RNA

Abstract

A simple method was established that allowed large quantities of human parainfluenza 3 (PF3) virions to be isolated from tissue culture cells. The purity of the virus was sufficient for biochemical analysis of virion proteins. The density of PF3 virions was 1.18-1.20. Purified virions contained seven viral proteins with estimated molecular weights of: L, 180 000; P, 83 000; HN, 69 000; NP, 66 000; F0, 60 000; F1, 51 000; and M, 38 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. There were three phosphoproteins, P, NP and M, and two glycoproteins, HN and F (includes F0 and F1). F1.2, the activated, cleaved, fusion glycoprotein (60 000 Da), consisting of two disulfide-linked subunits, F1 and F2, was seen only under nonreducing conditions. Because of its small size (approximately 9000 Da) F2 could be seen only on gels with high acrylamide concentrations. As in other enveloped viruses, cellular actin (43 000 Da) was present in purified virions. Several minor bands migrating between NP and M represented breakdown products of NP. Solubilization of the virion membrane in low salt buffer with non-ionic detergent resulted in the loss of HN and F. In high salt buffer, the M protein was also removed. Nucleocapsids isolated by CsCl centrifugation contained L, P, NP and small amounts of M. Nucleocapsids isolated in the presence of the ionic detergent, sarcosyl, contained only the NP protein. The density of nucleocapsids was 1.29-1.30. Genomic 50S RNA isolated from nucleocapsids had an estimated molecular weight of 5 X 10(6).