Changes in the translational landscape during red clover necrotic mosaic virus infection

ABSTRACT Viruses alter host gene expression to create a proviral environment, while the host simultaneously regulates gene expression to restrict viral spread. Owing to RNA viruses’ complete reliance on the host translational machinery, it is important to assess translational control during virus infection. Therefore, we used ribosome profiling (Ribo-seq) paired with RNA-seq to observe how red clover necrotic mosaic virus (RCNMV) infection of Arabidopsis plants alters cellular gene expression at the levels of mRNA abundance and translation efficiency. We determined that at 5 days post-inoculation (dpi), the translational response to RCNMV infection is enriched in genes of the innate immune system. Expression of a tumor necrosis factor receptor-associated factor (TRAF)-like protein, a regulator of development and immune response, was translationally but not transcriptionally upregulated early in systemic infection. By 8 dpi, many pathways were regulated/dysregulated, and unfolded protein response (UPR) genes were transcriptionally upregulated but with reduced translation efficiency. Ribosome profiling of RCNMV RNAs revealed (i) −1 programmed ribosomal frameshifting at 7.5%–8.0%, the first direct measurement of frameshift efficiency in infected cells for any plant virus; (ii) that coat protein is translated at extremely high efficiency, while the RNA-dependent RNA polymerase is translated least efficiently; and (iii) an unexpected extremely strong ribosomal pause site in the open reading frame that encodes the movement protein. To our knowledge, this is the first genome-wide study that assesses the translational control of gene expression in plants infected with a virus from the large and diverse Tombusviridae family. IMPORTANCE Positive-strand RNA viruses usurp the host’s translation machinery to synthesize viral proteins. Moreover, translation of host mRNAs is altered by virus infection, both as part of the host immune response and by the virus to inhibit host defenses. To assess all these changes globally, we used ribosome profiling of plants infected with a member of the large and ubiquitous Tombusviridae family. We identified key host genes and pathways that were differentially altered in translation efficiency, giving us an understanding of host responses not detectable by conventional RNA sequencing. Moreover, ribosome profiling revealed (i) the most accurate calculation of the efficiency of ribosomal frameshifting during infection for any plant virus, (ii) the extremely high level of translation of viral coat protein, and (iii) an unexpectedly strong ribosomal pause site in the movement protein gene. This work provides an understanding of a new dimension of gene expression control in plant-virus interactions.