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

Abstract

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 absent in the viral factories. This prompts the question of whether other poly(A)-binding proteins engage vaccinia virus post-replicative mRNA in viral factories. Here, 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) reduced vaccinia virus DNA replication, post-replicative protein levels, and viral production. Interestingly, the knockdown of LARP4 expression also reduced protein levels from transfected mRNA containing a 5′-poly(A) leader in vaccinia virus-infected and uninfected HFFs. Taken together, our results identified a poly(A)-binding protein, LARP4, being enriched in the vaccinia virus viral factories and facilitating viral replication in HFFs. IMPORTANCE 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), 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 reduced vaccinia virus DNA replication, post-replicative gene expression, and viral production.