title: Visualization of retrovirus uptake and delivery into acidic endosomes.
abstract: 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.
authors.affiliation: AIDS Research Center, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Miyauchi
authors.forename: Mariana
authors.initials: M
authors.lastname: Marin
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Melikyan
mesh_terms: D002460:Cell Line; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism / Q000821:virology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D014771:Virion / Q000235:genetics / Q000502:physiology; D053586:Virus Internalization
chemical_list: D049452:Green Fluorescent Proteins