Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

• Main Authors: de la Vega Michelle, Marin Mariana, Kondo Naoyuki, Miyauchi Kosuke, Kim Yuri, Epand Raquel F, Epand Richard M, Melikyan Gregory B
• Format: Article
• Language: English
• Published: BMC 2011-12-01
• Series: Retrovirology
• Subjects: HIV fusion kinetics; hemifusion; fusion pore; dynasore; temperature-arrested intermediate; fusion inhibitors; single particle tracking; beta-lactamase; intrinsic membrane curvature
• Online Access: http://www.retrovirology.com/content/8/1/99

<p>Abstract</p> <p>Background</p> <p>We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear.</p> <p>Results</p> <p>We examined HIV-1 fusion with a panel of target cells lines and with primary CD4⁺T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells <it>via </it>endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization.</p> <p>Conclusions</p> <p>Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.</p>