Characterization of HIV-1 Entry Site Specificity using Single-Particle Tracking

Abstract

The first critical step in the HIV-1 infectious cycle is fusion of the viral membrane with the membrane of the host cell and penetration of the viral capsid into the host cytosol. Because the functionality of the HIV-1 fusion glycoprotein (Env) is pH-independent, it was thought that productive HIV-1 fusion occurs at the plasma membrane (PM). This hypothesis is supported by the observations that HIV-1 mediates fusion between adjacent cells and that cell-cell fusion occurs between Env and receptor/co-receptor expressing cells. To the contrary, it was recently demonstrated that, after engaging receptor/co-receptor at the cell surface, HIV-1 traffics via an endocytic pathway before fusing with the endosomal membrane. However, the previous virus labeling techniques were unable to reliably detect fusion with the PM. Here, we directly quantified the fraction of HIV-1 virions that fuse with the PM by co-labeling viral particles with a pH-sensor incorporated into the viral membrane and a content marker that is released into the cytoplasm upon fusion. In imaging viruses bound to living cells, virus fusion at neutral pH is manifested as loss of the viral content marker without change to the signal from the pH-sensor. Upon virus entry to an acidic compartment, the reference signal from the pH-sensor is completely quenched, thus precluding detection of subsequent fusion. We found that only a small fraction of fusion events occur at neutral pH, presumably at the cell surface or in early, pH-neutral vesicles. Our finding implies that the majority of HIV-1 virions enters and fuses after trafficking into acidic compartments. Notably, the viral pH sensor also revealed occasional recycling of HIV-1 particles to the cell surface followed by their re-internalization. This work was supported by NIH R01 GM054787.