Bunyamwera orthobunyavirus S-segment untranslated regions mediate poly(A) tail-independent translation

Abstract

The mRNAs of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, possess a 5' cap structure but lack a 3' poly(A) tail, a common feature of eukaryotic mRNAs that greatly enhances translation efficiency. Viral mRNAs also contain untranslated regions (UTRs) that flank the coding sequence. Using model virus-like mRNAs that harbor the Renilla luciferase reporter gene, we found that the 3' UTR of the BUNV small-segment mRNA mediated efficient translation in the absence of a poly(A) tail. Viral UTRs did not increase RNA stability, and polyadenylation did not significantly enhance reporter activity. Translation of virus-like mRNAs in transfected cells was unaffected by knockdown of poly(A)-binding protein (PABP) but was markedly reduced by depletion of eukaryotic initiation factor 4G, suggesting a PABP-independent process for translation initiation. In BUNV-infected cells, translation of polyadenylated but not virus-like mRNAs was inhibited. Furthermore, we demonstrate that the viral nucleocapsid protein binds to, and colocalizes with, PABP in the cytoplasm early in infection, followed by nuclear retention of PABP. Our results suggest that BUNV corrupts PABP function in order to inhibit translation of polyadenylated cellular mRNAs while its own mRNAs are translated in a PABP-independent process.

FIG. 1.

Translation efficiency of nonpolyadenylated mRNAs. (A) Luciferase activity in BHK-21 cells. Cells were transfected with 0.5 μg in vitro-transcribed, 5′ capped but nonpolyadenylated mRNAs, and 18 h later, the cells were lysed and assayed for luciferase activity. The black circles represent 5′ cap structures. (B) Stability of reporter signals. BHK-21 cells were transfected as described above for panel A, and cells were lysed at 12-h intervals over the course of 48 h and assayed for luciferase activity. The error bars show the standard deviations from the means for three replicate experiments. (C) Stability of transfected mRNAs. Northern blot analysis of total RNA (2 μg/lane) extracted from transfected BHK-21 cells at the indicated time points (in hours after transfection). Renilla luciferase and actin mRNAs were detected with the respective digoxigenin-labeled probes. u, untransfected control.

FIG. 2.

Influence of polyadenylation on translation. (A) Agarose gel electrophoresis of in vitro-transcribed, nonpolyadenylated mRNA (−) and polyadenylated mRNA (+). m, size markers in bases. (B) Luciferase activity in transfected cells. BHK-21 cells were transfected with 0.5 μg of either nonpolyadenylated or polyadenylated (AAAA) transcripts as indicated and lysed 18 h later, and Renilla luciferase activity was determined. The error bars show the standard deviations from the means for three replicate experiments.

FIG. 3.

Translational activity resides within the 3′UTR. (A) Effect of omitting 3′ or 5′ UTR sequences on translation efficiency. BHK-21 cells were transfected with 0.5 μg of various capped, nonpolyadenylated mRNAs that either lack the complete 3′UTR (BUN-REN5′), the complete 5′UTR (BUN-REN3′861), or the 20 terminal nucleotides of the 3′UTR (BUN-REN3′841). The CRL-REN and BUN-REN RNAs were included as controls. Cells were lysed 18 h after transfection and assayed for Renilla luciferase activity. The black circles represent 5′ cap structures. The error bars show the standard deviations from the means for three replicate experiments. (B) Predicted secondary structure of the BUNV S-segment mRNA 3′UTR. The black lines at the bottom of the figure indicate the regions present in BUN-REN3′861 and BUN-REN3′841.

FIG. 4.

Translational requirement for PABP and eIF4G. (A) Effect of depletion of PABP or eIF4G.For the knockdown of PABP, HeLa cells were transfected with 60 pmol of either a PABP-specific siRNA (RNA interference [RNAi] PABP) or a nonsilencing mismatch siRNA (control). For the knockdown of eIF4G, cells were transfected with 1 μg of a plasmid that expresses the eIF4G-specific siRNA si31. Control cells were transfected with a plasmid encoding the mismatch siRNA si31M. After 48 h, the cells were transfected with 0.5 μg of the indicated mRNA. The cells were lysed 14 h later and assayed for Renilla luciferase activity. The average of two experiments is shown. (B) Western blot analysis of PABP and eIF4G expression. HeLa cells silenced as described in Materials and Methods were lysed 62 h after transfection, and proteins were separated by SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. The blots were probed for either PABP or eIF4G. The blots were also probed for actin and tubulin as loading controls.

FIG. 5.

Impact of virus infection on translation. BHK-21 cells were infected with either wt BUNV or rBUNVdelNSs2 at 5 PFU per cell or left uninfected (mock infected). After 9 h, the cells were transfected with 0.5 μg of the indicated mRNA, lysed 18 h later, and assayed for luciferase activity.

FIG. 6.

Interaction between BUNV and PABP. (A) PABP stability in infected cells. BHK-21 cells were infected with either wt BUNV or rBUNVdelNSs2 at 5 PFU per cell or mock infected as indicated. Cells were lysed 10 and 24 h postinfection (p.i.) and analyzed for expression of PABP, BUNV N, and tubulin by Western blotting. (B) Coimmunoprecipitation of N protein with cellular PABP. BHK-21 cells were infected with wt BUNV (wt) or rBUNdelNSs2 (del) or mock infected (m), and at 12 h postinfection, cell lysates were treated with RNase A (+) or not treated with RNase A (−), followed by immunoprecipitation (IP) with anti-PABP or anti-N protein antibodies. Precipitated proteins were analyzed by SDS-PAGE, followed by Western blotting (WB) and immunodetection with anti-PABP and anti-N antibodies as indicated.

FIG. 7.

Nuclear retention of PABP. BHK-21 cells were infected with wt BUNV or rBUNVdelNSs2 at 1 PFU per cell or mock infected. At 4, 8, and 22 h postinfection, the cells were fixed and analyzed by indirect immunofluorescence using antibodies to BUNV N (green) and PABP (red).