Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

Abstract

Sac(-) [murine Moloney sarcoma] cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with MW of 150,000, 60,000 and 23,000. The 60,000 and 23,000 MW polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000 MW protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000 and 23,000 MW polypeptides in messenger-dependent cell-free systems derived from mouse [fibroblast] L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000 MW polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000 and 23,000 MW in vitro products was established by comigration with virion proteins, immunoprecpitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNA which encoded p60 and p23 sedimented in sucrose-formamide gradients at 17S and 19S, respectively, and were clearly separable. These RNA were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA. The expression of the coronavirus JHM genome involves more than 1 subgenomic mRNA.