Ikeda Terumasa, Shimizu Ryo, Nasser Hesham, Carpenter Michael A, Cheng Adam Z, Brown William L, Sauter Daniel, Harris Reuben S
Division of Molecular Virology and Genetics, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto 8600811, Japan.
Graduate School of Medical Sciences, Kumamoto University, Kumamoto 8600811, Japan.
bioRxiv. 2023 Mar 29:2023.03.28.534666. doi: 10.1101/2023.03.28.534666.
HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4 T lymphocytes and macrophages. Previous studies have demonstrated that the APOBEC3 (A3) family of proteins (at least A3D, A3F, A3G, and stable A3H haplotypes) contribute to HIV-1 restriction in CD4 T lymphocytes. Virus-encoded virion infectivity factor (Vif) counteracts this antiviral activity by degrading A3 enzymes allowing HIV-1 replication in infected cells. In addition to A3 proteins, Vif also targets other cellular proteins in CD4 T lymphocytes, including PPP2R5 proteins. However, whether Vif primarily degrades only A3 proteins or has additional essential targets during viral replication is currently unknown. Herein, we describe the development and characterization of -, -, and -to- -null THP-1 cells. In comparison to Vif-proficient HIV-1, Vif-deficient viruses have substantially reduced infectivity in parental and -null THP-1 cells, and a more modest decrease in infectivity in -null cells. Remarkably, disruption of A3Aâ€"A3G protein expression completely restores the infectivity of Vif-deficient viruses in THP-1 cells. These results indicate that the primary function of Vif during HIV-1 replication in THP-1 cells is the targeting and degradation of A3 enzymes.
HIV-1 Vif neutralizes the HIV-1 restriction activity of A3 proteins. However, it is currently unclear whether Vif has additional essential cellular targets. To address this question, we disrupted to genes in the THP-1 myeloid cell line using CRISPR and compared the infectivity of wildtype HIV-1 and Vif mutants with the selective A3 neutralization activities. Our results demonstrate that the infectivity of Vif-deficient HIV-1 and the other Vif mutants is fully restored by ablating the expression of cellular A3A to A3G proteins. These results indicate that A3 proteins are the only essential target of Vif that is required for HIV-1 replication in THP-1 cells.
HIV-1必须克服多种先天性抗病毒机制才能在CD4 T淋巴细胞和巨噬细胞中复制。先前的研究表明，载脂蛋白B mRNA编辑酶催化多肽样3（APOBEC3，A3）蛋白家族（至少A3D、A3F、A3G和稳定的A3H单倍型）有助于在CD4 T淋巴细胞中限制HIV-1。病毒编码的病毒体感染性因子（Vif）通过降解A3酶来对抗这种抗病毒活性，从而使HIV-1在受感染细胞中复制。除A3蛋白外，Vif还靶向CD4 T淋巴细胞中的其他细胞蛋白，包括PPP2R5蛋白。然而，Vif在病毒复制过程中主要是仅降解A3蛋白还是有其他重要靶点目前尚不清楚。在此，我们描述了A3A、A3B和A3G基因敲除的THP-1细胞的构建及其特性。与具有Vif的HIV-1相比，缺乏Vif的病毒在亲本THP-1细胞和A3A基因敲除的THP-1细胞中的感染性大幅降低，而在A3B基因敲除细胞中的感染性下降幅度较小。值得注意的是，A3A至A3G蛋白表达的破坏完全恢复了THP-1细胞中缺乏Vif的病毒的感染性。这些结果表明，Vif在THP-1细胞中HIV-1复制过程中的主要功能是靶向和降解A3酶。
HIV-1 Vif中和A3蛋白的HIV-1限制活性。然而，目前尚不清楚Vif是否有其他重要的细胞靶点。为了解决这个问题，我们使用CRISPR在THP-1髓系细胞系中敲除A3A至A3G基因，并比较了野生型HIV-1和具有选择性A3中和活性的Vif突变体的感染性。我们的结果表明，通过消除细胞A3A至A3G蛋白的表达，缺乏Vif的HIV-1和其他Vif突变体的感染性完全恢复。这些结果表明，A3蛋白是Vif在THP-1细胞中HIV-1复制所需的唯一重要靶点。
