Visualization of retrovirus uptake and delivery into acidic endosomes

Diverse enveloped viruses enter cells by endocytosis and fusion with intracellular compartments. Recent evidence suggests that HIV also infects permissive cell lines by fusing with endosomes in a pH-independent manner. This finding highlights the importance of time-resolved monitoring of viral uptake. In the present study, we designed an imaging-based assay to measure endocytosis in real-time through probing the virus' accessibility to external solutions. Exposure of viruses bearing a pH-sensitive GFP (green fluorescent protein) variant on their surface to solutions of different acidity altered the fluorescence of surface-accessible particles, but not internalized viruses. By sequentially applying acidic and alkaline buffers with or without ammonium chloride, we were able to quantify the fractions of internalized and non-internalized virions, as well as the fraction of detached particles, over time. The exact time of single-virus internalization was assessed from the point when a particle ceased to respond to a perfusion with alternating acidic and alkaline buffers. We found that, surprisingly, HIV pseudoparticles entered acidic compartments shortly after internalization. These results suggest that the virus might be sorted to a quickly maturing pool of endocytic vesicles and thus be trafficked to fusion-permissive sites near the cell nucleus.