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High-speed imaging of ESCRT recruitment and dynamics during HIV virus like particle budding.

ABSTRACT: Endosomal sorting complexes required for transport proteins (ESCRT) catalyze the fission of cellular membranes during budding of membrane away from the cytosol. Here we have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the recruitment of ESCRTs specifically, ALIX, CHMP4b and VPS4 onto the budding HIV Gag virus-like particles (VLPs). We imaged the budding VLPs with 200 millisecond time resolution for 300 frames. Our data shows three phases for ESCRT dynamics: 1) recruitment in which subunits of ALIX, CHMP4b and VPS4 are recruited with constant proportions on the budding sites of HIV Gag virus like particles for nearly 10 seconds, followed by 2) disassembly of ALIX and CHMP4b while VPS4 signal remains constant for nearly 20 seconds followed by 3) disassembly of VPS4. We hypothesized that the disassembly observed in step 2 was catalyzed by VPS4 and powered by ATP hydrolysis. To test this hypothesis, we performed ATP depletion using (-) glucose medium, deoxyglucose and oligomycin. Imaging ATP depleted cells, we show that the disassembly of CHMP4b and ALIX observed in step 2 is ATP dependent. ATP depletion resulted in the recruitment of approximately 2-fold as many subunits of all ESCRTs. Resuming ATP production in cells, resulted in disassembly of the full ESCRT machinery which had been locked in place during ATP depletion. With some caveats, our experiments provide insight into the formation of the ESCRT machinery at the budding site of HIV during budding.

SUBMITTER: Gupta S

PROVIDER: S-EPMC7473513 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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High-speed imaging of ESCRT recruitment and dynamics during HIV virus like particle budding.

Gupta Shilpa S Bromley Josh J Saffarian Saveez S

PloS one 20200904 9

Endosomal sorting complexes required for transport proteins (ESCRT) catalyze the fission of cellular membranes during budding of membrane away from the cytosol. Here we have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the recruitment of ESCRTs specifically, ALIX, CHMP4b and VPS4 onto the budding HIV Gag virus-like particles (VLPs). We imaged the budding VLPs with 200 millisecond time resolution for 300 frames. Our data shows three phases for ESCRT dynamics: 1) recr ...[more]

PMID: 32886660

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Project description:BackgroundIt has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV.ResultsWe observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-alpha stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-alpha, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane.ConclusionOur study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.

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High-speed imaging of ESCRT recruitment and dynamics during HIV virus like particle budding.

Publications

High-speed imaging of ESCRT recruitment and dynamics during HIV virus like particle budding.

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