Analysis of rubella virus capsid protein-mediated enhancement of replicon replication and mutant rescue.
The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed deltaNotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (Cdelta8-Vero cells), a construct previously shown to be unable to complement DeltaNotI. In C-Vero cells but not Vero or Cdelta8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with deltaNotI (RUBrep/GFP-deltaNotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5' and 3' cis-acting elements were also rescued in C-Vero cells. Interestingly, the Cdelta8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in Cdelta8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.
Department of Biology, Georgia State University, 24 Peachtree Center Ave., Atlanta, Georgia 30303, USA.
Wen-Pin
WP
Tzeng
Jason D
JD
Matthews
Teryl K
TK
Frey
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D036022:Capsid Proteins / Q000502:physiology*; D002522:Chlorocebus aethiops; D009154:Mutation; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis*; D012093:Replicon; D012412:Rubella virus / Q000235:genetics*; D014709:Vero Cells
D020121:5' Untranslated Regions; D036022:Capsid Proteins; D012367:RNA, Viral