title: Visualization of Content Release from Cell Surface-Attached Single HIV-1 Particles Carrying an Extra-Viral Fluorescent pH-Sensor.
abstract: HIV-1 fusion leading to productive entry has long been thought to occur at the plasma membrane. However, our previous single virus imaging data imply that, after Env engagement of CD4 and coreceptors at the cell surface, the virus enters into and fuses with intracellular compartments. We were unable to reliably detect viral fusion at the plasma membrane. Here, we implement a novel virus labeling strategy that biases towards detection of virus fusion that occurs in a pH-neutral environment-at the plasma membrane or, possibly, in early pH-neutral vesicles. Virus particles are co-labeled with an intra-viral content marker, which is released upon fusion, and an extra-viral pH sensor consisting of ecliptic pHluorin fused to the transmembrane domain of ICAM-1. This sensor fully quenches upon virus trafficking to a mildly acidic compartment, thus precluding subsequent detection of viral content release. As an interesting secondary observation, the incorporation of the pH-sensor revealed that HIV-1 particles occasionally shuttle between neutral and acidic compartments in target cells expressing CD4, suggesting a small fraction of viral particles is recycled to the plasma membrane and re-internalized. By imaging viruses bound to living cells, we found that HIV-1 content release in neutral-pH environment was a rare event (~0.4% particles). Surprisingly, viral content release was not significantly reduced by fusion inhibitors, implying that content release was due to spontaneous formation of viral membrane defects occurring at the cell surface. We did not measure a significant occurrence of HIV-1 fusion at neutral pH above this defect-mediated background loss of content, suggesting that the pH sensor may destabilize the membrane of the HIV-1 pseudovirus and, thus, preclude reliable detection of single virus fusion events at neutral pH.
authors: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors: Chetan
authors: C
authors: Sood
authors: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors: Mariana
authors: M
authors: Marin
authors: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors: Caleb S
authors: CS
authors: Mason
authors: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, United States of America.|Children's Healthcare of Atlanta, Atlanta, Georgia, United States of America.
authors: Gregory B
authors: GB
authors: Melikyan
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism / Q000821:virology; D057809:HEK293 Cells; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018799:Intercellular Adhesion Molecule-1; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D013050:Spectrometry, Fluorescence; D014771:Virion / Q000502:physiology*; D053585:Virus Attachment; D053586:Virus Internalization; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D018799:Intercellular Adhesion Molecule-1; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria