Characterization of temperature-sensitive mutants of human respiratory syncytial (RS) virus

• Main Author: Caravokyri, Calliope
• Published: University of Warwick 1990
• Subjects: 579; QR Microbiology
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302799

Four ts mutants of RS virus, two from the subgroup A/wild-type strain A2 (ts A1 ts A2) and two from the subgroup B/wild-type strain RSN-2 (ts N1, ts N19), representing different complementation groups, were studied. Mutant ts A1 (complementation group A) was found to be restricted at a late stage of infection at 39°C. The degree of growth restriction was directly related to the length of incubation of ts A1-infected cultures at 39°C, implying that the spread of infection (by cell-to-cell fusion) was affected. The SDS-PAGE profile of radiolabelled ts A1 intracellular viral proteins at 39°C revealed a significant reduction in the amount of the F1 polypeptide (the large subunit of the fusion protein), followed by a slightly less severe decrease of the matrix (M) protein. The cleaved but undissociated fusion (F1,2) protein of ts A1 migrated more slowly than its wild-type counterpart on SDS-PAGE. Characterization of ts+ revertants revealed varying degrees of reversion to wild-type levels of growth at 39°C, but no coordinate restoration of the ts phenotype and of the ts A1 F1,2 was observed. This suggested that the mutation affecting F1,2 mobility in ts A1 could be compensated by secondary mutations in the same (F) or in another gene, or that the tsA1 lesion is located in a different viral protein with which the F protein forms a complex during the virus cycle. Mutant ts A2 (complementation group B) was found to exhibit temperature-sensitive synthesis of the G glycoprotein which mediates RS virus attachment to host cells. Examination of the G-processing pathway showed that synthesis of the major p50 precursor was unaffected but further maturation to fully glycosylated G was defective at 39°C. Mutant ts A2 also exhibited defective proteolytic cleavage of the Fo precursor into F1 and F2 subunits (a processing step required for creation of a fusion-active F protein) at both temperatures. This could explain the previously reported variability in plaque phenotype and growth of ts A2 under permissive conditions in different cell types, since the proteolytic activation of the fusion protein is host-dependent. Mutant ts N1 (complementation group D) possessed an M protein which disappeared from the soluble cytoplasmic fraction of infected cells soon after its synthesis at 39°C. The ts N1 M protein was also partially unstable at 33°C and exhibited slightly decreased SDS-PAGE mobility. The M protein of ts+ revertants was stable at 39°C, correlating the defect in M-stability with the ts N1 phenotype. The M proteins of three ts+ revertants exhibited the slower mobility of the ts N1 M protein, suggesting that they were pseudorevertants. The P protein of ts N1 (and its ts+ revertants) also migrated more slowly than the wild-type RSN-2 P protein. Sequence analysis and in vitro expression of a ts N1 P cDNA clone showed that the aberrant P mobility was due to a single amino acid change (Asn → Asp at position 217). Mutant ts N19 (complementation group E) was completely restricted at 39°C, indicative of a ts defect at an early stage of infection. The ts N19 P protein lacked an epitope recognized by anti-P MAb 3-5. The P proteins of ts+ revertants, which exhibited wild-type growth at 39°C, had this epitope restored, confirming that loss of the P-epitope was correlated with the ts phenotype. Sequence determination of P cDNA clones, produced from RSN-2, ts N19 and ts+ revertant P mRNA by reverse transcription and PCR amplification, identified a single amino acid difference (Gly → Ser at position 172) in the ts N19 P protein. In vitro expression of the sequenced P cDNA clones and immunoprecipitation of the respective P proteins with MAb 3-5 showed that presence of the Gly172 residue was required for MAb- binding. This residue was contained within a C-terminal domain of the P protein which could be independently expressed by internal initiation of in vitro translation at AUG148.