The polypeptides of human respiratory syncytial virus: Products of cell-free protein synthesis and post-translational modifications

Abstract

The properties and inter-relationships of virus-induced polypeptides synthesized in BS-C-1 cells infected with human respiratory syncytial (RS) virus have been investigated in vivo and in vitro. Analysis of the kinetics of synthesis of RS virus-induced polypeptides in vivo provided no evidence for temporal controls, a situation comparable to that observed with other negative-stranded RNA viruses with unsegmented genomes. In vitro translation of cytoplasmic RNA extracted from infected cells resulted in the synthesis of six virus-induced polypeptides, four of which were of similar molecular weights to species produced in infected cells. Synthesis of all of these polypeptides was directed by polyA)-containing RNA. The identity of the major nucleocapsid polypeptide (VP 41) synthesized in vitro was confirmed by peptide mapping. A prominent polypeptide in infected cells, VP 38, was not synthesized in vitro. The relationship of this polypeptide to other RS virus polypeptides was investigated in detail, and it was observed that VP 38 and VP 41 exhibited a precursor-product relationship in pulse-chase experiments and appeared to be related by peptide mapping. However, addition of the protease inhibitors tolylsulfonyl-lysyl chloromethylketone (TLCK) and tolylsulfonyl-phenylalanyl chloromethylketone to infected cells during labeling enhanced the recovery of VP 41 and correspondingly decreased VP 38. A similar effect was obtained if addition of TLCK was delayed until the time of cell lysis. Taken together, these observations suggest that during the course of infection, the nucleocapsid polypeptide VP 41 undergoes a transition from a protease-sensitive form (detected by the occurrence of VP 38) to a protease-resistant form. Two other hitherto unreported post-translational modifications of RS virus-induced polypeptides, sulfation and phosphorylation, are described.