Fate of minus-strand templates and replication complexes produced by a p23-cleavage-defective mutant of Sindbis virus.
SIN2V is an engineered mutant Sindbis virus (SIN) that is unable to process the P23 cleavage site in polyproteins P123 and P1234 that are translated from the genome after its entry into cells. Unlike wild-type (wt) SIN, it caused minus strands to be made continuously and replication-transcription complex (RTC) activity to be unstable (R. Gorchakov, E. Frolova, S. Sawicki, S. Atasheva, D. Sawicki, and I. Frolov, J. Virol. 82:6218-6231, 2008). We examined further the effects of P23 on SIN RNA replication and RTC activity. Continuous minus-strand synthesis by SIN2V produced 250% of wt levels of minus strands but accumulated only 110% of wt levels (0.39 pg, or 2.7 x 10(4) molecules of double-stranded RNA per cell). Because SIN2V-infected cells accumulated only 40% of the minus strands that were made, cells must possess some process to limit RTC accumulation. The loss of activity by SIN2V RTC after translation was inhibited was stochastic and not due to their inherent instability, based on finding that activity was lost without the degradation of the minus-strand templates. In addition to their normal functions, P23 RTCs exhibited the novel phenotype of being unable to switch from making less to making more genomes than subgenomic 26S mRNA at late times during infections. Our results lend credence to the hypothesis that nsP2 (and possibly nsP3) possesses functions other than those needed solely for RTC activity and that it may also act with the host to regulate minus-strand synthesis and the stability of the RTC.
Mai
Junbo
J
Department of Medical Microbiology and Immunology, University of Toledo College of Medicine, Health Science Campus, 3000 Arlington Avenue, Toledo, OH 43614, USA.
Sawicki
Stanley G
SG
Sawicki
Dorothea L
DL
D003546:Cysteine Endopeptidases / Q000502:physiology*; D008954:Models, Biological; D020815:Polyproteins / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020871:RNA Stability; D012367:RNA, Viral / Q000378:metabolism*; D012845:Sindbis Virus / Q000235:genetics / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D014779:Virus Replication
D020815:Polyproteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C089383:nsp3 protein, alphavirus; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase