The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment

Abstract

The positive-strand RNA genomes of caliciviruses are not capped, but are instead covalently linked at their 5' ends to a viral protein called VPg. The lack of a cap structure typical of eukaryotic mRNA and absence of an internal ribosomal entry site suggest that VPg may function in translation initiation on calicivirus RNA. This hypothesis was tested by analyzing binding of Norwalk virus VPg to translation initiation factors. The eIF3d subunit of eIF3 was identified as a binding partner of VPg by yeast two-hybrid analysis. VPg bound to purified mammalian eIF3 and to eIF3 in mammalian cell lysates. To test the effects of the VPg- eIF3 interaction on translation, VPg was added to cell-free translation reactions programmed with either capped reporter RNA, an RNA containing an EMCV internal ribosomal entry site (IRES) or an RNA with a cricket paralysis virus IRES. VPg inhibited translation of all reporter RNAs in a dose-dependent manner. Together, the data suggest that VPg may play a role in initiating translation on calicivirus RNA through unique protein-protein interactions with the translation machinery.

Fig. 1. VPg binds initiation factor eIF3. Pull-down assays were conducted with GST or GST–VPg immobilized on glutathione–Sepharose 4B beads. (A) Pull-down with purified eIF3. The input eIF3 lane represents 10% of the amount of protein used in the pull-down reaction, and the western blot was probed with anti-eIF3 polyclonal antibody. (B) Pull-down with ³⁵S-labeled in vitro translated subunit eIF3d. The input eIF3d lane represents 25% of the amount used in the GST and GST–VPg pull-downs. Pull-down eluates were separated by 10% SDS–PAGE and exposed to film for autoradiography.

Fig. 2. VPg binds eIF3 present in cell lysates. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. (A) CaCo-2 cell lysates. (B) S7-treated CaCo-2 lysates. (C) GST–SMV-VPg immobilized to glutathione–Sepharose beads. (D) VPg deletion mutants. Western blots were probed with anti-eIF3 polyclonal antibody. Asterisks indicate a protein likely to be eIF4GI.

Fig. 3. VPg interactions with eIF4GI and other initiation factors. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. Western blots were probed with (A) anti-eIF4GI, or (B) anti-eIF2α, anti-eIF4E, anti-S6 or anti-eIF4B.

Fig. 4. VPg inhibits translation of reporter RNAs. (A) m⁷G-Luc RNA, (B) IRES-Luc RNA, and (C) CrPV IGR-IRES RNA were translated in Flexi RRL without or with RNA (lanes 1 and 2) and with increasing concentrations of GST–VPg (lanes 3, 4 and 5 = 12, 24 and 48 pmol, respectively), or increasing concentrations of GST (lanes 6, 7 and 8 = 19, 38 and 76 pmol, respectively). ³⁵S-labeled proteins were separated by SDS–10% PAGE and visualized by autoradiography. (D) Average OD of luciferase synthesized in the presence of GST–VPg in the translation reactions shown in (A–C). m⁷G-Luc RNA (squares), IRES-Luc (triangles) and CrPV IGR-IRES (crosses). A control reaction where GST was used as the inhibitor in the IRES-Luc translation reactions is shown as a representative (diamonds). Data are normalized to 1.