Ribosome-mediated specificity in vesicular stomatitis virus mRNA translation defines a new role for rpL40 during initiation

Vesicular stomatitis virus (VSV) infection causes inhibition of host protein synthesis, in part by sequestering initiation factors required for mRNA cap recognition. The viral mRNAs share a common mRNA structure to those of the host cell, with a 5' cap and 3' polyadenylate tail, but continue to be efficiently translated despite host translational shutoff. This observation suggests that a non-canonical translation pathway is utilized for viral protein synthesis. To investigate this pathway, we performed an RNA interference screen to identify genes required for VSV replication. In contrast to bulk cellular translation, viral translation is hypersensitive to knockdown of a protein constituent of the 60S ribosomal subunit, rpL40. Depletion of rpL40 diminishes VSV protein synthesis by >90% and is restored through complementation with an siRNA-resistant mutant of rpL40. To delineate the mechanism by which rpL40 is required for viral protein synthesis, we reconstituted translation of VSV mRNA in yeast extracts in vitro. In the absence of rpL40, we show that the two ribosomal subunits fail to associate on VSV mRNA, and the small subunit does not scan to the initiation codon. Regulation by rpL40 occurs in context of the large subunit, providing direct evidence for translational control by the ribosome itself. This rpL40- dependent mechanism of translation initiation is broadly conserved within eukaryotes, governed solely through an RNA determinant, and is utilized by several viruses within the order Mononegavirales. To determine whether a subset of cellular transcripts also require rpL40 for translation, we identified polysome-associated mRNAs in yeast by deep sequencing. We demonstrate that in vitro and in vivo translation of candidate mRNAs, including factors involved in stress responses, are inhibited in the absence of rpL40. This finding suggests that rpL40 plays a critical role in transcript-specific translation during cellular stress. Collectively, our work identifies an alternative translation pathway that is specifically dependent on rpL40, revealing a previously unappreciated mechanism of protein synthesis regulation by the ribosome.