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. 2014 Mar 7;6(3):1078-90.
doi: 10.3390/v6031078.

Evidence showing that tetraspanins inhibit HIV-1-induced cell-cell fusion at a post-hemifusion stage

Menelaos Symeonides  1 , Marie Lambelé  2 , Nathan H Roy  1 , Markus Thali  3
Affiliations

Evidence showing that tetraspanins inhibit HIV-1-induced cell-cell fusion at a post-hemifusion stage

Menelaos Symeonides et al. Viruses. .

Abstract

Human immunodeficiency virus type 1 (HIV-1) transmission takes place primarily through cell-cell contacts known as virological synapses. Formation of these transient adhesions between infected and uninfected cells can lead to transmission of viral particles followed by separation of the cells. Alternatively, the cells can fuse, thus forming a syncytium. Tetraspanins, small scaffolding proteins that are enriched in HIV-1 virions and actively recruited to viral assembly sites, have been found to negatively regulate HIV-1 Env-induced cell-cell fusion. How these transmembrane proteins inhibit membrane fusion, however, is currently not known. As a first step towards elucidating the mechanism of fusion repression by tetraspanins, e.g., CD9 and CD63, we sought to identify the stage of the fusion process during which they operate. Using a chemical epistasis approach, four fusion inhibitors were employed in tandem with CD9 overexpression. Cells overexpressing CD9 were found to be sensitized to inhibitors targeting the pre-hairpin and hemifusion intermediates, while they were desensitized to an inhibitor of the pore expansion stage. Together with the results of a microscopy-based dye transfer assay, which revealed CD9- and CD63-induced hemifusion arrest, our investigations strongly suggest that tetraspanins block HIV-1-induced cell-cell fusion at the transition from hemifusion to pore opening.

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Figures

Figure 1
Figure 1
Schematic of fusion steps, and stage of action of fusion inhibitors used, denoted as numbered red circles: (1) AMD3100; (2) C34; (3) Imatinib; and (4) dynasore. Schematic key: Outer membrane leaflets–blue; Inner membrane leaflets–green; HIV-1 Env gp120–yellow, gp41–brown (cytoplasmic tail not shown); CD4–orange; CXCR4–grey; Tetraspanins–red.
Figure 2
Figure 2
Chemical epistasis approach; possible outcomes of tandem tetraspanin overexpression with fusion inhibitor titration.
Figure 3
Figure 3
Chemical epistasis experiments using fusion inhibitors AMD3100 (A); C34 (B); Imatinib (C); and dynasore (D) in virus-producing HeLa cells overexpressing L6 or CD9 co-cultured with TZM-bl target cells. Fusion levels were quantified using the DSP fusion assay. The basal effect of L6 and CD9 overexpression on fusion (shown here as an inset in B; data taken from the same experiment) was equalized by normalizing to vehicle. The data points were fitted to a classical inhibition response model and the inhibitor concentrations leading to 25% inhibition (IC25) or 50% inhibition (IC50) were compared between L6 and CD9 for each inhibitor. Shown are the only comparisons which exhibited a significant difference.
Figure 4
Figure 4
Accumulation of the hemifusion intermediate caused by tetraspanin overexpression. (A) HeLa cells transfected with pNL4-3Gag−iGFP and the indicated plasmids were co-cultured with CMAC and CM-DiI-labeled Jurkat cells, fixed, and imaged. The three possible outcomes of cell-cell contact are shown as examples. Inset in Hemifusion/CM-DiI panel is contrast-enhanced. Bar = 10 μm; (B) Quantification of full fusion. The number of CMAC-positive nuclei found within GFP-positive syncytia was divided by the number of GFP-positive cells and normalized to the empty vector control; (C) Quantification of hemifusion (cells positive for GFP and CM-DiI, but not CMAC) as a percentage of the sum of hemifusions and syncytia; (D) Total fusion activity measured as the sum of hemifusions and syncytia, normalized to the empty vector control. Statistical comparisons made against L6, except where denoted by a horizontal bar.
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