La-related protein 4 is enriched in vaccinia virus factories and is required for efficient viral replication in primary human fibroblasts

Abstract

In addition to the 3-poly(A) tail, vaccinia virus mRNAs synthesized after viral DNA replication (post-replicative mRNAs) possess a 5-poly(A) leader that confers a translational advantage in virally infected cells. These mRNAs are synthesized in viral factories, the cytoplasmic compartment where vaccinia virus DNA replication, mRNA synthesis, and translation occur. However, a previous study indicates that the poly(A)-binding protein (PABPC1)-which has a well-established role in RNA stability and translation-is not present in the viral factories. This prompts the question of whether another poly(A)-binding protein engages vaccinia virus post-replicative mRNA in viral factories. In this study, we found that La-related protein 4 (LARP4), a poly(A) binding protein, was enriched in viral factories in multiple types of cells during vaccinia virus infection. Further studies showed that LARP4 enrichment in the viral factories required viral post-replicative gene expression and functional decapping enzymes encoded by vaccinia virus. We further showed that knockdown of LARP4 expression in human foreskin fibroblasts (HFFs) significantly reduced vaccinia virus post-replicative gene expression and viral replication. Interestingly, the knockdown of LARP4 expression also reduced 5-poly(A) leader-mediated mRNA translation in vaccinia virus-infected and uninfected HFFs. Together, our results identified a poly(A)-binding protein, LARP4, enriched in the vaccinia virus viral factories and facilitates viral replication and mRNA translation. ImportancePoxviruses are a family of large DNA viruses comprising members infecting a broad range of hosts, including many animals and humans. Poxvirus infections can cause deadly diseases in humans and animals. Vaccinia virus, the prototype poxvirus, encodes over 200 open reading frames (ORFs). Over 90 of vaccinia virus ORFs are transcribed post-viral DNA replication. All these mRNAs contain a 5-poly(A) leader, as well as a 3-poly(A) tail. They are synthesized in viral factories, where vaccinia virus DNA replication, mRNA synthesis and translation occur. However, surprisingly, the poly(A) binding protein (PABPC1) that is important for mRNA metabolism and translation is not present in the viral factories, suggesting other poly(A) binding protein(s) may be present in viral factories. Here we found another poly(A)-binding protein, La-related protein 4 (LARP4), is enriched in viral factories during vaccinia virus infection. We also showed that LARP4 enrichment in the viral factories depends on viral post-replicative gene expression and functional viral decapping enzymes. The knockdown of LARP4 expression in human foreskin fibroblasts (HFFs) significantly reduced vaccinia virus post-replicative gene expression and viral replication. Overall, this study identified a poly(A)-binding protein that plays an important role in vaccinia virus replication.