Section of Infections of the Nervous System.
Flow and Imaging Cytometry Core Facility.
AIDS. 2020 Jun 1;34(7):963-978. doi: 10.1097/QAD.0000000000002512.
Astrocytes are proposed to be a critical reservoir of HIV in the brain. However, HIV infection of astrocytes is inefficient in vitro except for cell-to-cell transmission from HIV-infected cells. Here, we explore mechanisms by which cell-free HIV bypasses entry and postentry barriers leading to a productive infection.
HIV infection of astrocytes was investigated by a variety of techniques including transfection of CD4-expressing plasmid, treatment with lysosomotropic agents or using a transwell culture system loaded with HIV-infected lymphocytes. Infection was monitored by HIV-1 p24 in culture supernatants and integrated proviral DNA was quantified by Alu-PCR.
Persistent HIV infection could be established in astrocytes by transfection of proviral DNA, transduction with VSV-G-pseudotyped viruses, transient expression of CD4 followed by HIV infection, or simultaneous treatment with lysosomotropic chloroquine or Tat-HA2 peptide with HIV infection. In absence of these treatments, HIV entered via endocytosis as seen by electronmicroscopy and underwent lysosomal degradation without proviral integration, indicating endocytosis is a dead end for HIV in astrocytes. Nevertheless, productive infection was observed when astrocytes were in close proximity but physically separated from HIV-infected lymphocytes in the transwell cultures. This occurred with X4 or dual tropic R5X4 viruses and was blocked by an antibody or antagonist to CXCR4.
A CD4-independent, CXCR4-dependent mechanism of viral entry is proposed, by which immature HIV particles from infected lymphocytes might directly bind to CXCR4 on astrocytes and trigger virus--cell fusion during or after the process of viral maturation. This mechanism may contribute to the formation of brain HIV reservoirs.
星形胶质细胞被认为是大脑中 HIV 的重要储存库。然而，除了从 HIV 感染细胞的细胞间传播外，星形胶质细胞中 HIV 的感染效率很低。在这里，我们探讨了无细胞 HIV 绕过进入和进入后屏障从而导致有效感染的机制。
通过多种技术研究了 HIV 对星形胶质细胞的感染，包括转染表达 CD4 的质粒、用溶酶体趋向性药物处理或使用加载有 HIV 感染淋巴细胞的 Transwell 培养系统。通过培养上清液中的 HIV-1 p24 监测感染，并用 Alu-PCR 定量整合的前病毒 DNA。
通过转染前病毒 DNA、用 VSV-G 假型病毒转导、短暂表达 CD4 后进行 HIV 感染、或同时用溶酶体趋向性氯喹或 Tat-HA2 肽处理与 HIV 感染，可在星形胶质细胞中建立持续的 HIV 感染。在没有这些处理的情况下，HIV 通过内吞作用进入细胞，如电子显微镜所见，并在没有前病毒整合的情况下发生溶酶体降解，表明内吞作用是 HIV 在星形胶质细胞中的死胡同。然而，当星形胶质细胞在 Transwell 培养中与感染 HIV 的淋巴细胞接近但物理分离时，观察到了有效的感染。这种情况发生在 X4 或双嗜性 R5X4 病毒中，并且可以被 CXCR4 的抗体或拮抗剂阻断。
提出了一种 CD4 非依赖性、CXCR4 依赖性的病毒进入机制，在此机制下，来自感染淋巴细胞的不成熟 HIV 颗粒可能直接与星形胶质细胞上的 CXCR4 结合，并在病毒成熟过程中或之后触发病毒-细胞融合。这种机制可能有助于形成大脑 HIV 储存库。
