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Journal of Virology, June 1999, p. 5176-5180, Vol. 73, No. 6
Laboratory of Infectious Diseases, National
Institute of Allergy and Infectious Diseases, Bethesda, Maryland
20892-0720
Received 28 October 1998/Accepted 17 February 1999
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
0022-538X/99/$04.00+0
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
Leu and Met-1169Val, 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°C for RNA replication, mRNA
synthesis, and virus growth. The 1009 virus was partially restricted
for RNA synthesis and virus growth even at 32°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.
*
Corresponding author. Mailing address: Laboratory of
Infectious Diseases, National Institute of Allergy and Infectious
Diseases, 7 Center Dr. MSC 0720, Bethesda, MD 20892-0720. Phone: (301)
496-3481. Fax: (301) 496-8312. E-mail:
pcollins{at}atlas.niaid.nih.gov.
Journal of Virology, June 1999, p. 5176-5180, Vol. 73, No. 6
0022-538X/99/$04.00+0
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