Analysis of rubella virus capsid protein-mediated enhancement of replicon replication and mutant rescue

Abstract

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.

FIG. 1.

RUBrep/CAT-ΔNotI expression in Vero cell lines expressing C protein. (Top) Diagram of the RUB genome and the region of the C gene used to generate Vero cell lines that stably express aa residues 1 to 277 (C-Vero) or 9 to 277 (CΔ8-Vero) of the RUB C protein. Both constructs contained a tandem FLAG epitope at the N terminus. The location in the genome of the NotI region, the insertion site of the HA epitope in RUBrep-HA/GFP, the 5′ UTR, and the 3′ CAE are also shown. (Bottom) Vero cells, C-Vero cells, and CΔ8-Vero cells were transfected with RUBrep/CAT-ΔNotI replicon transcripts. At 3 days posttransfection, the cells were lysed and CAT activity assayed. Mock-transfected Vero cells were included as a background control. The results shown are the averages from three independent experiments.

FIG. 2.

GFP expression and RNA synthesis by wt and mutant RUBrep/GFP in Vero cells and Vero cell lines expressing C protein. (A) Vero cells, C-Vero cells, and CΔ8-Vero cells were transfected with RUBrep/GFP, RUBrep/GFP-ΔNotI, or RUBrep/GFP-GDD* transcripts and monitored daily for GFP expression. Micrographs taken on days 2 and 4 posttransfection are shown. (B) Vero cells, C-Vero cells, and CΔ8-Vero cells were transfected with transcripts from RUBrep/GFP constructs with the following 3′ CAE mutations: ΔSL1, ΔSL2, ΔSL3, or ΔSL4. At 2 days posttransfection, total cell RNA was isolated and assayed by Northern blotting for the accumulation of replicon-specific RNA species (G, genomic RNA). (C) C-Vero and Vero cells were transfected with RUBrep/GFP transcripts, and total cell RNA was isolated at 1, 2, 3, and 4 days posttransfection and assayed for the accumulation of replicon-specific RNAs.

FIG. 3.

Subcellular localization of C protein in Vero cell lines stably expressing C protein constructs. Vero (A and E), C-Vero (B and F), CΔ8-Vero cells (C and G), and Vero cells infected with Robo502 virus (multiplicity of infection, 5 PFU/cell; fixed 48 h postinfection) (D and H) were costained with an anti-C (αC) monoclonal antibody (micrographs made using the same exposure) (A to D) and Hoechst 33342 (micrographs made using the same exposure) (E to H).

FIG. 4.

P150 accumulation in cells transfected with replicon transcripts. Vero and C-Vero cells were mock transfected or transfected with RUBrep-HA/GFP, RUBrep-HA/GFP-ΔSL1, RUBrep-HA/GFP-D3, RUBrep-HA/GFP-GDD*, or RUBrep-HA/GFP-ΔNotI transcripts. Lysates made 6 (A) and 24 (B) hours posttransfection were resolved by SDS-PAGE, followed by Western blotting. The blot was divided in three horizontally, and the appropriate strips were probed with anti-HA (α-HA) antibody to detect HA-tagged P150, with anticalnexin (α-calnexin) antibody to detect calnexin as an internal control, or with anti-GFP (α-GFP) antibody to detect GFP synthesized from replicons under control of the subgenomic RNA.

FIG. 5.

Coimmunoprecipitation of P150 and C in RUBrep-HA/GFP-transfected C-Vero cells. C-Vero cells mock transfected or transfected with RUBrep-HA/GFP transcripts were lysed at 3 days posttransfection. The lysates were immunoprecipitated (IP) with either anti-HA (α-HA) antibody (to immunoprecipitate HA-tagged P150) or anti-FLAG (α-FLAG) antibody (to immunoprecipitate FLAG-tagged C). Following SDS-PAGE and Western blotting (WB), the blots were probed with either anti-HA antibody or anti-FLAG antibody. HA-tagged P150 coimmunoprecipitating with FLAG-tagged C and FLAG-tagged C coimmunoprecipitating with HA-tagged P150 are marked with asterisks.