Center for Immunotherapy, Vaccines, and Virotherapy Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA.
J Virol. 2021 Jun 24;95(14):e0015121. doi: 10.1128/JVI.00151-21.
RNA helicase A/DHX9 is required for diverse RNA-related essential cellular functions and antiviral responses and is hijacked by RNA viruses to support their replication. Here, we show that during the late replication stage in human cancer cells of myxoma virus (MYXV), a member of the double-stranded DNA (dsDNA) poxvirus family that is being developed as an oncolytic virus, DHX9, forms unique granular cytoplasmic structures, which we named "DHX9 antiviral granules." These DHX9 antiviral granules are not formed if MYXV DNA replication and/or late protein synthesis is blocked. When formed, DHX9 antiviral granules significantly reduced nascent protein synthesis in the MYXV-infected cancer cells. MYXV late gene transcription and translation were also significantly compromised, particularly in nonpermissive or semipermissive human cancer cells where MYXV replication is partly or completely restricted. Directed knockdown of DHX9 significantly enhanced viral late protein synthesis and progeny virus formation in normally restrictive cancer cells. We further demonstrate that DHX9 is not a component of the canonical cellular stress granules. DHX9 antiviral granules are induced by MYXV, and other poxviruses, in human cells and are associated with other known cellular components of stress granules, dsRNA and virus encoded dsRNA-binding protein M029, a known interactor with DHX9. Thus, DHX9 antiviral granules function by hijacking poxviral elements needed for the cytoplasmic viral replication factories. These results demonstrate a novel antiviral function for DHX9 that is recruited from the nucleus into the cytoplasm, and this step can be exploited to enhance oncolytic virotherapy against the subset of human cancer cells that normally restrict MYXV. The cellular DHX9 has both proviral and antiviral roles against diverse RNA and DNA viruses. In this article, we demonstrate that DHX9 can form unique antiviral granules in the cytoplasm during myxoma virus (MYXV) replication in human cancer cells. These antiviral granules sequester viral proteins and reduce viral late protein synthesis and thus regulate MYXV, and other poxviruses, that replicate in the cytoplasm. In addition, we show that in the absence of DHX9, the formation of DHX9 antiviral granules can be inhibited, which significantly enhanced oncolytic MYXV replication in human cancer cell lines where the virus is normally restricted. Our results also show that DHX9 antiviral granules are formed after viral infection but not by common nonviral cellular stress inducers. Thus, our study suggests that DHX9 has antiviral activity in human cancer cells, and this pathway can be targeted for enhanced activity of oncolytic poxviruses against even restrictive cancer cells.
RNA 解旋酶 A/DHX9 是多种 RNA 相关基本细胞功能和抗病毒反应所必需的，并且被 RNA 病毒劫持以支持其复制。在这里，我们表明，在双链 DNA（dsDNA）痘病毒家族成员兔粘液瘤病毒（MYXV）的晚期复制阶段，DHX9 在人类癌细胞中形成独特的细胞质颗粒结构，我们将其命名为“DHX9 抗病毒颗粒”。如果 MYXV DNA 复制和/或晚期蛋白合成被阻断，则不会形成这些 DHX9 抗病毒颗粒。当形成时，DHX9 抗病毒颗粒会显著减少 MYXV 感染的癌细胞中的新生蛋白合成。MYXV 晚期基因的转录和翻译也受到显著影响，特别是在非许可或半许可的人类癌细胞中，MYXV 复制受到部分或完全限制。DHX9 的定向敲低显著增强了正常限制性癌细胞中的病毒晚期蛋白合成和病毒后代的形成。我们进一步证明 DHX9 不是细胞应激颗粒的组成部分。DHX9 抗病毒颗粒由 MYXV 和其他痘病毒在人细胞中诱导产生，与应激颗粒的其他已知细胞成分、dsRNA 和病毒编码的 dsRNA 结合蛋白 M029 相关，后者是与 DHX9 已知的相互作用蛋白。因此，DHX9 抗病毒颗粒通过劫持痘病毒细胞质复制工厂所需的元素来发挥作用。这些结果表明，DHX9 具有一种新的抗病毒功能，该功能从细胞核被募集到细胞质中，并且可以利用这一步骤来增强针对通常限制 MYXV 的人类癌细胞亚群的溶瘤病毒治疗。细胞 DHX9 对多种 RNA 和 DNA 病毒具有促病毒和抗病毒作用。在本文中，我们证明在 MYXV 复制过程中，DHX9 可以在人类癌细胞的细胞质中形成独特的抗病毒颗粒。这些抗病毒颗粒隔离病毒蛋白并减少病毒晚期蛋白合成，从而调节 MYXV 和其他在细胞质中复制的痘病毒。此外，我们表明，在缺乏 DHX9 的情况下，DHX9 抗病毒颗粒的形成可以被抑制，这显著增强了在病毒通常受限的人类癌细胞系中的溶瘤 MYXV 复制。我们的研究结果还表明，DHX9 抗病毒颗粒是在病毒感染后形成的，而不是由常见的非病毒细胞应激诱导剂形成的。因此，我们的研究表明，DHX9 在人类癌细胞中具有抗病毒活性，并且可以针对甚至是限制性的癌细胞，靶向该途径以增强溶瘤痘病毒的活性。
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