Significance HIV-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The assembly of virus particles involves numerous host and viral proteins that are potential therapeutic targets. We used high-resolution microscopy techniques to investigate how the virus hijacks cellular proteins to enable the release of virions from an infected cell. We show with high temporal and spatial resolution that components of the host endosomal sorting complex required for transport (ESCRT) machinery are recruited to the neck of the assembling virus to facilitate scission of the link between the virus and the cell.

The Endosomal Sorting Complexes Required for Transport III (ESCRT-III) proteins are critical for cellular membrane scission processes with topologies inverted relative to clathrin-mediated endocytosis. Some viruses appropriate ESCRT-IIIs for their release. By imaging single assembling viral-like particles of HIV-1, we observed that ESCRT-IIIs and the ATPase VPS4 arrive after most of the virion membrane is bent, linger for tens of seconds, and depart ~20 s before scission. These observations suggest that ESCRT-IIIs are recruited by a combination of membrane curvature and the late domains of the HIV-1 Gag protein. ESCRT-IIIs may pull the neck into a narrower form but must leave to allow scission. If scission does not occur within minutes of ESCRT departure, ESCRT-IIIs and VPS4 are recruited again. This mechanistic insight is likely relevant for other ESCRT-dependent scission processes including cell division, endosome tubulation, multivesicular body and nuclear envelope formation, and secretion of exosomes and ectosomes.

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HIV-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

7 clathrin-mediated endocytosis. Some viruses appropriate ESCRT-IIIs for their release. By 8 imaging single assembling viral-like particles of HIV-1, we observed that ESCRT-IIIs and 9 the ATPase VPS4 arrive after most of the virion membrane is bent, linger for tens of 10 seconds, and depart ~20 seconds before scission. These observations suggest ESCRT-IIIs 11 are recruited by a combination of membrane curvature and the late domains of the HIV-1 12 Gag protein. ESCRT-IIIs may pull the neck into a narrower form but must leave to allow 13 scission. If scission does not occur within minutes of ESCRT departure, ESCRT-III and 14 VPS4 are recruited again. This mechanistic insight is likely relevant for other ESCRT 15 dependent scission processes including cell division, endosome tubulation, multivesicular 16 body and nuclear envelope formation, and secretion of exosomes and ectosomes. 17 18