The major attenuating mutations of the respiratory syncytial virus vaccine candidate cpts530/1009 specify temperature-sensitive defects in transcription and replication and a non-temperature-sensitive alteration in mRNA termination.
The live-attenuated respiratory syncytial virus vaccine candidate cpts530/1009 was previously shown to contain two separate amino acid changes in the L protein, mutations 530 and 1009 (Phe-521-->Leu and Met-1169-->Val, respectively, according to the amino acid sequence of the L protein). Each mutation independently specifies temperature-sensitive (ts) and attenuation phenotypes. In this study, we examined the effects of these mutations on transcription and RNA replication, using complete infectious recombinant virus as well as a plasmid-based minireplicon system, the latter under conditions in which effects on replication and transcription are uncoupled. In comparison with recombinant wild-type virus, the 530 and 1009 viruses were partially restricted at 37 degrees C for RNA replication, mRNA synthesis, and virus growth. The 1009 virus was partially restricted for RNA synthesis and virus growth even at 32 degrees C, which suggested that the 1009 mutation has a non-ts component in addition to the ts component. Interestingly, the synthesis of polycistronic readthrough mRNAs was elevated 1.6- to 3.8-fold for the 1009 virus, and this defect was non-ts. Studies with the minigenome system showed that the 530 and 1009 mutations each directly affect both replication and transcription, that the effect on replication was marginally greater than on transcription for the 530 mutation, and that the increase in readthrough mRNA associated with the 1009 mutation also was observed with the minigenome system.
Juhasz
K
K
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0720, USA.
Murphy
B R
BR
Collins
P L
PL
D009154:Mutation; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D012136:Respiratory Syncytial Viruses / Q000235:genetics*; D013696:Temperature; D014158:Transcription, Genetic; D014613:Vaccines, Attenuated; D014765:Viral Vaccines
D012333:RNA, Messenger; D012367:RNA, Viral; D014613:Vaccines, Attenuated; D014765:Viral Vaccines