Tryptic peptide analysis of the structural proteins of a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Abstract

The individual structural polypeptides of HVJ (haemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) were examined by tryptic peptide analysis. [3H]-methionine-labelled structural proteins of the wild-type virus of HVJ (HVJ-W) and [35S]-methionine-labelled corresponding constituent proteins of a temperature-sensitive (ts) mutant (HVJ-pB) derived from an HVJ carrier culture were compared by ion-exchange chromatography on columns of P-type chromobeads. The tryptic peptides of the individual structural proteins showed characteristic elution profiles. In all the structural proteins tested, the chromatographic elution profiles of both strains generally showed a close resemblance. However, certain minor peaks which were present in one strain but absent in the other strain were detected in the preparations of the P, HN, and F polypeptides. Further, analysis of the NP polypeptide showed that a major peak of one strain appeared at a position in the pH gradient different from a seemingly corresponding major peak of the other strain. In the M protein some possibly homologous minor peaks were found to differ between the two strains.

Isolation and Characterization of Temperature‐Sensitive Mutants of Sendai Virus

“…The former were divided into seven complementation groups (22). Among these 11 ts mutants, however, only a few were characterized in terms of defective protein (9,11,21,23,24). We isolated 16 ts mutants from mutagenized virus stocks as well as persistently infected cultures, i.e., carrier cultures, and analyzed these mutants genetically, biochemically and biologically.…”

Temperature-sensitive HVJ (Sendai Virus) with Altered P Polypeptide Derived from Persistently Infected Cell Lines

“…Wechsler et al (1979) noted that virus persistently infected cells might undergo an extensive genetic change since point mutations usually were not reflected as alterations in electrophoretic mobility of proteins. Kimura (1979) was able to detect some differences in tryptic peptide analysis of the M protein between an M defective ts mutant with slow mobility of the M protein and the wild-type HVJ, although we found no differences between normal (HVJo) and altered P proteins by partial protease digestion. However, it is still unclear what kind of altered functions of M protein occur as a result of its variation in tool.…”

Search for Sendai 6/94 viral RNA in the antigen-free cell line Cl-C-2 isolated from human multiple sclerosis brain tissue

“…Reverse transcriptase reaction. Purified 6/94 50S RNA from egg-grown virus was incubated in an assay (100 p.1) containing 5 mM magnesium acetate, 90 mM KCl, 10 mM DTE, 50 mM Tris-hydrochloride (pH 8.3), 1 mM ATP, 20 ,ug of actinomycin D per ml, 200 ,ug of bovine serum albumin per ml, dATP, dCTP, and dTTP of 0.33 mM each, 20 ,uCi of [3H]dGTP (13 Ci/mmol; Amersham Buchler), 100 U of reverse transcriptase (RT) from avian myeloblastosis virus per ml, 10 pg of oligodeoxyguanidylic acid [oligo(dG)] per ml, and 100 p.g of 6/94 RNA per ml. The mixture was incubated at 42°C for 30 min, and the nucleic acid was extracted with phenol and precipitated with ethanol.…”

“…Several mechanisms possibly responsible for the induction and maintenance of persistent viral infections are under discussion. In cell cultures persistently infected by paramyxoviruses like Sendai virus, the factors mainly involved seem to be (i) ts mutants (12,14,15,24,26), (ii) defective interfering particles (4,27,28), (iii) interferon (2, 6-11), and (iv) an alteration of viral proteins, particularly of the M protein (13,14,17,32).…”