Dhungel Pragyesh, Brahim Belhaouari Djamal, Yang Zhilong
Division of Biology, Kansas State University , Manhattan, Kansas, USA.
Department of Veterinary Pathobiology, School of Veterinary Medicine & Biomedical Sciences, Texas A&M University , College Station, Texas, USA.
Microbiol Spectr. 2023 Aug 18;11(5):e0139023. doi: 10.1128/spectrum.01390-23.
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.
除了 3' - 聚腺苷酸尾外，痘苗病毒 DNA 复制后合成的 mRNA（复制后 mRNA）还具有 5' - 聚腺苷酸前导序列，该序列在病毒感染的细胞中赋予翻译优势。这些 mRNA 在病毒工厂中合成，病毒工厂是痘苗病毒 DNA 复制、mRNA 合成和翻译发生的细胞质区室。然而，先前的一项研究表明，在 RNA 稳定性和翻译中具有明确作用的聚腺苷酸结合蛋白（PABPC1）在病毒工厂中不存在。这就引发了一个问题，即是否有其他聚腺苷酸结合蛋白在病毒工厂中与痘苗病毒复制后 mRNA 相互作用。在本研究中，我们发现聚腺苷酸结合蛋白 La 相关蛋白 4（LARP4）在痘苗病毒感染期间在多种类型细胞的病毒工厂中富集。进一步的研究表明，LARP4 在病毒工厂中的富集需要病毒复制后基因表达和痘苗病毒编码的功能性脱帽酶。我们还表明，在人包皮成纤维细胞（HFFs）中敲低 LARP4 的表达会降低痘苗病毒 DNA 复制、复制后蛋白水平和病毒产量。有趣的是，在痘苗病毒感染和未感染的 HFFs 中，敲低 LARP4 的表达也会降低含有 5' - 聚腺苷酸前导序列的转染 mRNA 的蛋白水平。综上所述，我们的结果鉴定出一种聚腺苷酸结合蛋白 LARP4，它在痘苗病毒工厂中富集并促进 HFFs 中的病毒复制。重要性痘苗病毒是痘病毒的原型，编码超过 200 个开放阅读框（ORF）。超过 90 个痘苗病毒 ORF 在病毒 DNA 复制后转录。所有这些 mRNA 都含有 5' - 聚腺苷酸前导序列以及 3' - 聚腺苷酸尾。它们在病毒工厂中合成，痘苗病毒 DNA 复制、mRNA 合成和翻译在此发生。然而，令人惊讶的是，对 mRNA 代谢和翻译很重要的聚腺苷酸结合蛋白 PABPC1 在病毒工厂中不存在，这表明可能有其他聚腺苷酸结合蛋白存在于病毒工厂中。在这里，我们发现另一种聚腺苷酸结合蛋白 La 相关蛋白 4（LARP4）在痘苗病毒感染期间在病毒工厂中富集。我们还表明，LARP4 在病毒工厂中的富集取决于病毒复制后基因表达和功能性病毒脱帽酶。在人包皮成纤维细胞中敲低 LARP4 的表达会降低痘苗病毒 DNA 复制、复制后基因表达和病毒产量。
