Date Published: June 21, 2019
Publisher: Public Library of Science
Author(s): William J. Neidermyer, Sean P. J. Whelan, Peter Sarnow.
Infection of mammalian cells with vesicular stomatitis virus (VSV) results in the inhibition of cellular translation while viral translation proceeds efficiently. VSV RNA synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces 5 mRNAs that are structurally indistinct to cellular mRNAs with respect to their 5′ cap-structure and 3′-polyadenylate tail. Using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mRNA, we interrogate the impact of VSV infection of HeLa cells on translation. Analysis of sequence reads in the different fractions shows >60% of total cytoplasmic and polysome-associated reads map to the 5 viral genes by 6 hours post-infection, a time point at which robust host cell translational shut-off is observed. Consistent with an overwhelming abundance of viral mRNA in the polysome fraction, the reads mapping to cellular genes were reduced. The cellular mRNAs that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more AU rich, features that are shared with the viral mRNAs. Several of those mRNAs encode proteins known to positively affect viral replication, and using chemical inhibition and siRNA depletion we confirm that the host chaperone heat shock protein 90 (hsp90) and eukaryotic translation initiation factor 3A (eIF3A)—encoded by 2 such mRNAs—support viral replication. Correspondingly, regulated in development and DNA damage 1 (Redd1) encoded by a host mRNA with reduced polysome association inhibits viral infection. These data underscore the importance of viral mRNA abundance in the shut-off of host translation in VSV infected cells and link the differential translatability of some cellular mRNAs with pro- or antiviral function.
Infection of mammalian cells by vesicular stomatitis virus (VSV) results in a profound shut-off of host cell gene expression. This host cell shut-off occurs at the level of mRNA transcription through inhibition of RNA polymerase II by the viral-encoded matrix protein (M) [1–3]. The M protein also forms a complex with ribonucleic acid export 1 (Rae1) and nucleoporin 98 (Nup98)  thus suppressing host cell mRNP export from the nucleus, including that of mature cellular mRNAs [5–8]. VSV infection also inhibits protein synthesis by manipulation of the host-cell translation machinery, particularly at the level of translation initiation [9, 10]. Eukaryotic initiation factor 4E (eIF4E)—the rate limiting factor for translation initiation—recognizes the 7mGpppN mRNA cap structure as part of the eIF4F complex, and in concert with other translation initiation factors facilitates the recruitment of the small 40S ribosomal subunit to the mRNA prior to scanning to the initiating methionine where the 60S subunit joins [11, 12]. VSV infection results in the rapid dephosphorylation of eIF4E itself, for which the functional consequences are unclear, and of its binding protein (eIF4E-BP1) leading to eIF4E sequestration and the suppression of translation initiation [9, 10].
We obtained two snapshots into the complex battle for control of protein synthesis in cells infected with vesicular stomatitis virus by sequencing of polysome-associated mRNAs at 2 and 6 hpi. Those snapshots provide further evidence that the abundance of vesicular stomatitis virus mRNAs is a determinant of the dominance of viral protein synthesis in infected cells, but highlight several additional attributes of this complex relationship. Those include the demonstration that some host mRNAs that remain polysome associated encode proteins that support viral replication, and some of those that exhibit reduced polysome association encode proteins that are antiviral. We also obtained further insight into the seemingly paradoxical observations that viral infection results in a reduction of the available pool of eIF4E –the rate limiting factor for translation initiation–yet viral mRNAs contain a cap structure that is indistinct to that of host mRNAs. Through the use of a viral mutant defective in mRNA cap methylation, and suppression of eIF4E levels we provide evidence consistent with a transition from an eIF4E dependent phase of viral translation to one less-dependent on eIF4E.