Single-Particle Tracking of HIV-1 Virions Bearing an Extra-Viral Fluorescent pH Sensor Reveals Viral Entry occurs after Trafficking to an Acidic Cellular Compartment

Abstract

The HIV-1 infectious cycle begins with viral glycoprotein mediated fusion with the host cell membrane and subsequent entry of the viral capsid to the host cytosol. It has been proposed that HIV-1 fusion and productive entry occur at the plasma membrane (PM) because functionality of the HIV-1 fusion glycoprotein (Env) is pH-independent. Further supporting this hypothesis are the observations that HIV-1 mediates fusion between adjacent cells and that cell-cell fusion occurs between Env- and receptor/co-receptor-bearing cells. However, it was recently demonstrated that, after Env engagement of receptor/co-receptor at the cell surface, the virus traffics via an endocytic pathway before fusing with the endosomal membrane. Though convincing in its conclusion, this study was unable to reliably detect viral fusion at the PM. Here, we implement a virus labeling strategy that biases towards detection of virus fusion that occurs in a pH-neutral environment, at the PM or, possibly, in early pH-neutral vesicles. Virus particles are co-labeled with an intra-viral content-marker released upon viral fusion and an extra-viral pH sensor that fully quenches upon trafficking to a mildly acidic compartment, precluding subsequent detection of viral content release. In imaging viruses bound to living cells, we found that HIV-1 content release in neutral-pH environment was a rare event with efficiency not significantly greater than the background content-loss that occurs due to non-specific increases in viral membrane permeability. Our finding implies that the majority of HIV-1 entry events occur after the virus particle traffics to an acidic cellular compartment. Interestingly, this labeling strategy also reveals that HIV-1 particles occasionally shuttle between neutral and acidic compartments, suggesting that the viral particles interact with a cellular recycling pathway. This work supported by NIH R01 GM054787.