Herold Nikolas, Anders-Ößwein Maria, Glass Bärbel, Eckhardt Manon, Müller Barbara, Kräusslich Hans-Georg
Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany
J Virol. 2014 Dec;88(24):13956-70. doi: 10.1128/JVI.01543-14. Epub 2014 Sep 24.
Cytoplasmic entry of HIV-1 requires binding of the viral glycoproteins to the cellular receptor and coreceptor, leading to fusion of viral and cellular membranes. Early studies suggested that productive HIV-1 infection occurs by direct fusion at the plasma membrane. Endocytotic uptake of HIV-1 was frequently observed but was considered to constitute an unspecific dead-end pathway. More recent evidence suggested that endocytosis contributes to productive HIV-1 entry and may even represent the predominant or exclusive route of infection. We have analyzed HIV-1 binding, endocytosis, cytoplasmic entry, and infection in T-cell lines and in primary CD4(+) T cells. Efficient cell binding and endocytosis required viral glycoproteins and CD4, but not the coreceptor. The contribution of endocytosis to cytoplasmic entry and infection was assessed by two strategies: (i) expression of dominant negative dynamin-2 was measured and was found to efficiently block HIV-1 endocytosis but to not affect fusion or productive infection. (ii) Making use of the fact that HIV-1 fusion is blocked at temperatures below 23 °C, cells were incubated with HIV-1 at 22 °C for various times, and endocytosis was quantified by parallel analysis of transferrin and fluorescent HIV-1 uptake. Subsequently, entry at the plasma membrane was blocked by high concentrations of the peptidic fusion inhibitor T-20, which does not reach previously endocytosed particles. HIV-1 infection was scored after cells were shifted to 37 °C in the presence of T-20. These experiments revealed that productive HIV-1 entry occurs predominantly at the plasma membrane in SupT1-R5, CEM-ss, and primary CD4(+) T cells, with little, if any, contribution coming from endocytosed virions.
HIV-1, like all enveloped viruses, reaches the cytoplasm by fusion of the viral and cellular membranes. Many viruses enter the cytoplasm by endosomal uptake and fusion from the endosome, while cell entry can also occur by direct fusion at the plasma membrane in some cases. Conflicting evidence regarding the site of HIV-1 fusion has been reported, with some studies claiming that fusion occurs predominantly at the plasma membrane, while others have suggested predominant or even exclusive fusion from the endosome. We have revisited HIV-1 entry using a T-cell line that exhibits HIV-1 endocytosis dependent on the viral glycoproteins and the cellular CD4 receptor; results with this cell line were confirmed for another T-cell line and primary CD4(+) T cells. Our studies show that fusion and productive entry occur predominantly at the plasma membrane, and we conclude that endocytosis is dispensable for HIV-1 infectivity in these T-cell lines and in primary CD4(+) T cells.
HIV-1进入细胞质需要病毒糖蛋白与细胞受体及共受体结合，从而导致病毒膜与细胞膜融合。早期研究表明，有活性的HIV-1感染是通过在质膜处直接融合发生的。HIV-1的内吞摄取经常被观察到，但被认为是一条非特异性的死胡同途径。最近的证据表明，内吞作用有助于有活性的HIV-1进入，甚至可能是主要或唯一的感染途径。我们分析了HIV-1在T细胞系和原代CD4⁺T细胞中的结合、内吞、细胞质进入及感染情况。有效的细胞结合和内吞需要病毒糖蛋白和CD4，但不需要共受体。通过两种策略评估了内吞作用对细胞质进入和感染的贡献：（i）检测了显性负性发动蛋白-2的表达，发现其能有效阻断HIV-1内吞，但不影响融合或有活性的感染。（ii）利用HIV-1融合在23℃以下温度被阻断这一事实，将细胞在22℃与HIV-1孵育不同时间，通过对转铁蛋白和荧光HIV-1摄取的平行分析对内吞作用进行定量。随后，高浓度的肽类融合抑制剂T-20阻断质膜处的进入，T-20不会到达先前已内吞的颗粒。在T-20存在的情况下将细胞转移至37℃后对HIV-1感染进行评分。这些实验表明，在SupT1-R5、CEM-ss和原代CD4⁺T细胞中，有活性的HIV-1进入主要发生在质膜，内吞的病毒颗粒即使有贡献也很小。
HIV-1与所有包膜病毒一样，通过病毒膜与细胞膜融合进入细胞质。许多病毒通过内体摄取并从内体融合进入细胞质，而在某些情况下细胞进入也可通过在质膜处直接融合发生。关于HIV-1融合位点的证据相互矛盾，一些研究称融合主要发生在质膜，而另一些研究则表明主要或甚至唯一的融合发生在内体。我们使用一种依赖病毒糖蛋白和细胞CD4受体表现出HIV-1内吞作用的T细胞系重新研究了HIV-1进入；该细胞系的结果在另一个T细胞系和原代CD4⁺T细胞中得到了证实。我们的研究表明融合和有活性的进入主要发生在质膜，并且我们得出结论，在这些T细胞系和原代CD4⁺T细胞中，内吞作用对于HIV-1感染性是不必要的。
