HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institutegrid.48336.3a, Frederick, Maryland, USA.
Biological Sciences, University of Denver, Denver, Colorado, USA.
J Virol. 2022 Dec 14;96(23):e0087622. doi: 10.1128/jvi.00876-22. Epub 2022 Nov 10.
The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that small interfering RNA (siRNA)-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR-Cas9 technology was used to knock out the expression of this protein in several human T-cell lines-Jurkat E6.1, SupT1, and H9-and in primary human CD4 T cells. knockout caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T cells. In H9 cells, knockout caused a cell density-dependent defect in virus replication. In primary CD4 T cells, knockout had no effect on HIV-1 replication. Furthermore, human T-cell leukemia virus type 1 (HTLV-1)-transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail (YW) implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. The incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types-human T-cell lines and primary CD4 T cells-we used CRISPR-Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4 T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4 T cells.
HIV-1 包膜糖蛋白（Env）包含一个长的细胞质尾巴，其中包含高度保守的基序，这些基序指导 Env 的运输和掺入病毒，并促进病毒的有效传播。细胞运输因子 Rab11a 家族相互作用蛋白 1C（FIP1C）已被牵连到 Env 向病毒组装部位的定向运输中。在这项研究中，我们证实，用小干扰 RNA（siRNA）介导的 FIP1C 耗尽可适度降低 HeLa 细胞中 Env 的掺入病毒。为了确定 FIP1C 是否需要在生理相关细胞中进行 Env 掺入和 HIV-1 复制，我们使用 CRISPR-Cas9 技术敲除了几种人 T 细胞系-Jurkat E6.1、SupT1 和 H9-以及原代人 CD4 T 细胞中的该蛋白的表达。在 SupT1 细胞中，FIP1C 的缺失导致 Env 掺入适度减少，但不抑制 SupT1 或 Jurkat E6.1 T 细胞中的病毒复制。在 H9 细胞中，FIP1C 的缺失导致病毒复制密度依赖性缺陷。在原代 CD4 T 细胞中，FIP1C 的缺失对 HIV-1 复制没有影响。此外，允许 HIV-1 复制的人类 T 细胞白血病病毒 1（HTLV-1）转化细胞系不表达 FIP1C。在 SupT1、Jurkat E6.1、H9 和原代 T 细胞中，Env 细胞质尾巴中涉及 FIP1C 介导的 Env 掺入的芳香基基序的突变会损害病毒复制，而与 FIP1C 的表达无关。这些结果表明，虽然 FIP1C 可能在某些情况下有助于 HIV-1 Env 的掺入，但对于 T 细胞中的 Env 掺入和病毒传播，可能需要其他潜在的冗余宿主因子。HIV-1 包膜（Env）糖蛋白 gp120 和 gp41 掺入病毒颗粒对于病毒感染性至关重要。gp41 包含一个长的细胞质尾巴，据推测它与包括运输因子 Rab11a 家族相互作用蛋白 1C（FIP1C）在内的宿主细胞因子相互作用。为了研究 FIP1C 在相关细胞类型-人 T 细胞系和原代 CD4 T 细胞中的作用，我们使用 CRISPR-Cas9 敲除了 FIP1C 的表达，并研究了对 HIV-1 Env 掺入和病毒复制的影响。我们观察到，在两种被研究的 T 细胞系（Jurkat E6.1 和 SupT1）和原代 CD4 T 细胞中，FIP1C 缺失不破坏 HIV-1 复制，而 FIP1C 缺失降低了 H9 细胞中的 Env 表达并延迟了复制。结果表明，虽然 FIP1C 可能在某些细胞系中有助于 Env 的掺入，但它不是原代 CD4 T 细胞中有效复制 HIV-1 所必需的。
