Infection of cells by human immunodeficiency virus type 1 (HIV-1) is a multistep process beginning with the sequential binding of the gp120 subunit of the viral envelope glycoprotein (Env) to CD4 and a coreceptor CCR5 or CXCR4 [1, 2]. Interactions with CD4 and coreceptor trigger conformational changes in the transmembrane subunit of Env, gp41, which ultimately mediates membrane fusion [3, 4]. As with other viruses that do not depend on low pH for entry, HIV-1 has been widely believed to undergo fusion at the plasma membrane, whereas endocytosis has been regarded as a nonproductive pathway leading to virus degradation (for example, [5-7]). This view is based mainly on the following lines of evidence. First, HIV-1 Env mediates cell-cell fusion at neutral pH [7, 8], and the virus itself can fuse adjacent cells expressing CD4 and coreceptors [9, 10] (termed “fusion from without”). Second, HIV-1 infection is not compromised by mutations in the cytoplasmic domains of CD4 or coreceptors that severely impair their ability to undergo ligand-mediated endocytosis [5, 6, 11, 12]. Third, in contrast to HIV-1, VSV G-pseudotyped HIV particles, which constitutively enter through endocytosis, exhibit different requirements for HIV-1 accessory proteins for infection [13], and strikingly, fail to infect resting CD4+ T cells [14-16]. Also, the membrane transport activity of Arf6 (ADP-ribosylation factor 6) appears essential for clathrin-independent CD4/HIV-1 co-internalization and fusion, but not for fusion of VSV G pseudotypes [17].
The above evidence, while supporting HIV-1 fusion with the plasma membrane, are somewhat indirect and generally do not rule out the existence of an endocytic entry pathway for this virus. For instance, the lack of low pH-dependence [8, 18, 19] simply means that HIV-1 fusion is not restricted to acidic compartments. It also remains to be demonstrated that CD4 and coreceptor mutants impaired in ligand-mediated endocytosis do not co-internalize with the virus, which would allow fusion with endosomes. On the other hand, accumulating evidence support the existence of productive HIV-1 entry through endocytosis. The observation that trans-dominant negative mutants of dynamin-2 and Eps15 potently inhibit HIV-1 fusion and infection [20] implies that this virus relies, at least in part, on clathrin-mediated endocytosis for productive entry. Moreover, a specific small-molecule inhibitor of clathrin function interferes with HIV-1 uptake and infectivity [21]. Finally, inhibition of dynamin GTPase activity by dynasore effectively suppressed clathrin-dependent uptake of transferrin and low density lipoprotein [22], as well as HIV-1 endocytosis, fusion and infectivity [23].
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By employing non-invasive imaging technologies and functional assays, we have gained further insights into the mechanism of HIV-1 entry [23]. First, visualization of single virus entry into cells revealed two types of fusion events – transfer of the viral lipids into the plasma membrane without the subsequent release of the viral content (hemifusion) and release of the viral content without significant dilution of the viral membrane marker, which corresponds to complete virus fusion with small intracellular compartments. Second, comparison of the rates of HIV-1 escape from a membrane-impermeable fusion inhibitor and from low temperature applied at different times during the virus entry demonstrated a delayed protection from the temperature block compared to resistance to a fusion inhibitor. Collectively, these findings imply that HIV-1 fuses with endosomes, but fails to undergo complete fusion with the plasma membrane. We also found that endosomal fusion was inhibited by a dynamin-2 inhibitor, dynasore, suggesting that dynamin is involved both in the virus uptake and in the subsequent fusion step occurring within endosomes [23].
The basis for HIV’s strong preference for entry through endosomes is not clear. Contrary to the model proposed in [24], kinetic measurements of lipid mixing with the plasma membrane demonstrated that the lack of complete fusion at the cell surface was not due to the faster virus uptake which would clear the virus from the plasma membrane before it undergoes fusion. Nearly 80% of Env- and receptor-dependent lipid mixing events at the plasma membrane occurred before significant endocytosis or endosomal fusion were detected [23, 25]. Here, to gain further insight into the determinants of HIV-1 fusion, we tested whether this virus was able to fuse with the plasma membrane of distinct target cells, such as lymphoid cells and U87.CD4.CCR5 cells, which appeared to support limited fusion between immobilized viruses and the plasma membrane of detached cells [26]. Moreover, we attempted to redirect HIV-1 fusion to the cell surface by preventing virus uptake, using endocytosis inhibitors or reduced temperature. However, these interventions did not favor complete fusion at the cell surface, in spite of the extended window of opportunity to enter from the plasma membrane. Our results thus reinforce the notion that HIV-1 enters different cell types by endocytosis and demonstrate that the failure of this virus to fuse with the plasma membrane is not due to the kinetically dominant endocytosis that removes fusogenic viruses from the cell surface. Finally, we found that dynasore blocked productive endocytosis of HIV-1 more efficiently than virus-endosome fusion, consistent with a more critical role of dynamin in endocytic vesicle formation compared to virus-endosome fusion.
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