Selective protein synthesis during vaccinia virus-induced host shutoff

Abstract

During infection, poxvirus makes host cells conducive for viral replication by causing host shutoff that is marked by global inhibition of host protein synthesis. Host shutoff facilitates the reallocation of cellular resources for viral replication and evasion of host antiviral immune responses. However, it poses a challenge for continuous synthesis of crucial cellular proteins and viral proteins that are important for viral replication. It is unclear whether and how viral and specific cellular proteins are selectively synthesized during poxvirus-induced host shutoff. In this dissertation, we elucidated that vaccinia virus boosts viral post-replicative protein synthesis by using the 5'-poly(A) leader at the 5'-UTR. Vaccinia virus has evolutionarily optimized the length of the poly(A) leader, and uninterrupted poly(A) leader is required for promoting poxvirus protein production. During vaccinia virus-induced shutoff, poly(A) leader stipulates viral post-replicative mRNAs an adaptive mechanism to translate efficiently. The poly(A) leader translation was not mediated by an internal ribosome entry site (IRES) mode, albeit poly(A) leader mediates cap-independent mode of translation. Through further investigation, we uncovered a cellular RNA-binding protein La-Related Protein 4 (LARP4) that was repurposed to augment vaccinia virus post-replicative mRNA translation. During VACV infection, LARP4 is enriched in the virus factory where VACV post-replicative mRNAs are translated. A decrease of LARP4 protein level reduces VACV replication, blocks post-replicative protein synthesis, and decreases 5'-poly(A) leader mediated translational advantage. Further studies showed that LARP4 is vital for the cap-independent mode of translation from poly(A) leader. We also showed that infection of vaccinia virus, the prototypic poxvirus, induced selective synthesis of cellular proteins involved in oxidative phosphorylation. Using simultaneous RNA-seq and ribosome profiling, we determined the mRNAs encoding proteins for oxidative phosphorylation complexes had increased relative translation efficiency. Indeed, vaccinia virus infection increased the activity of oxidative phosphorylation. Inhibition of oxidative phosphorylation function suppressed vaccinia virus replication significantly. Moreover, the mRNAs of oxidative phosphorylation have short 5'-UTRs with a less complex secondary structure that could confer oxidative phosphorylation mRNAs a translational advantage in vaccinia virus-infected cells during host shutoff. Together, these studies advanced our understanding of how vaccinia virus selectively synthesizes viral and cellular proteins for efficient viral replication during host shutoff. The findings may facilitate the development of novel anti-poxvirus strategies and the improvement of poxviruses as vaccine vectors and anti-cancer agents.