Nigos Lance R, Scott Nichollas E, Brooks Andrew G, Ait-Goughoulte Malika, Londrigan Sarah L, Reading Patrick C, Farrukee Rubaiyea
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne, VIC 3000, Australia.
Roche Pharma Research and Early Development, Roche Innovation Center Basel, 4070 Basel, Switzerland.
Pathogens. 2023 Jun 20;12(6):852. doi: 10.3390/pathogens12060852.
Host cell restriction factors are intracellular proteins that can inhibit virus replication. Characterisation of novel host cell restriction factors can provide potential targets for host-directed therapies. In this study, we aimed to assess a member of the Tripartite-motif family protein (TRIM) family, TRIM16, as a putative host cell restriction factor. To this end, we utilized constitutive or doxycycline-inducible systems to overexpress TRIM16 in HEK293T epithelial cells and then tested for its ability to inhibit growth by a range of RNA and DNA viruses. In HEK293T cells, overexpression of TRIM16 resulted in potent inhibition of multiple viruses, however, when TRIM16 was overexpressed in other epithelial cell lines (A549, Hela, or Hep2), virus inhibition was not observed. When investigating the antiviral activity of endogenous TRIM16, we report that siRNA-mediated knockdown of TRIM16 in A549 cells also modulated the mRNA expression of other TRIM proteins, complicating the interpretation of results using this method. Therefore, we used CRISPR/Cas9 editing to knockout TRIM16 in A549 cells and demonstrate that endogenous TRIM16 did not mediate antiviral activity against the viruses tested. Thus, while initial overexpression in HEK293T cells suggested that TRIM16 was a host cell restriction factor, alternative approaches did not validate these findings. These studies highlight the importance of multiple complementary experimental approaches, including overexpression analysis in multiple cell lines and investigation of the endogenous protein, when defining host cell restriction factors with novel antiviral activity.
宿主细胞限制因子是能够抑制病毒复制的细胞内蛋白质。鉴定新型宿主细胞限制因子可为宿主导向疗法提供潜在靶点。在本研究中，我们旨在评估三方基序家族蛋白（TRIM）家族的成员TRIM16作为一种假定的宿主细胞限制因子。为此，我们利用组成型或强力霉素诱导系统在HEK293T上皮细胞中过表达TRIM16，然后测试其抑制多种RNA和DNA病毒生长的能力。在HEK293T细胞中，TRIM16的过表达导致对多种病毒的有效抑制，然而，当TRIM16在其他上皮细胞系（A549、Hela或Hep2）中过表达时，未观察到病毒抑制作用。在研究内源性TRIM16的抗病毒活性时，我们报告在A549细胞中，siRNA介导的TRIM16敲低也调节了其他TRIM蛋白的mRNA表达，使得使用该方法解释结果变得复杂。因此，我们使用CRISPR/Cas9编辑技术在A549细胞中敲除TRIM16，并证明内源性TRIM16并未介导针对所测试病毒的抗病毒活性。因此，虽然最初在HEK293T细胞中的过表达表明TRIM16是一种宿主细胞限制因子，但其他方法并未证实这些发现。这些研究强调了多种互补实验方法的重要性，包括在多个细胞系中进行过表达分析以及对内源性蛋白质的研究，在定义具有新型抗病毒活性的宿主细胞限制因子时。
