Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion

Abstract

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically-encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 virions were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells.

Fluorescence Microscopy of the HIV-1 Envelope

“…For a detailed review on imaging HIV entry, uncoating, and nuclear entry, the reader could refer to the manuscript by Francis and Melikyan [39]. Instead, fluorescent imaging approaches have used the lipophilic dye DiD to localise HIV virions and resolve different entry steps [40][41][42][43][44][45][46]. When combined with a second intraviral marker (e.g., the Viral Protein R (Vpr) labelled with EGFP), the fluorescence signal from membrane dyes may be employed to report on viral envelope fusion with cell membranes [40,42].…”

“…In the case of plasma membrane entry, the membrane signal is diluted in the immense volume of cell membranes upon fusion, as opposed to the intraviral marker that stays trapped in the core and is finally released to the cytosol [41,42]. On the other hand, during endosomal entry, the membrane dye is not diluted due to the small volume of endosomes, but the intraviral marker signal disappears after capsid release to the cytosol [42,46]. More precisely, DiD fluorescence signal lost reports on hemifusion, i.e., the merger of the external leaflets of both membranes [43].…”

“…Some commercial setups are readily implementing fluorescence lifetime imaging microscopy (FLIM) for spatially-resolved measurement of the fluorescence lifetime of fluorescent tags. Lifetime can provide additional information about the structure or environment of a fluorescent molecule and has successfully been employed in the HIV [44,46] and membrane biophysics [20] research fields. Not only established microscopy techniques are being refined, but also new ones are being developed.…”

“…Advances in fluorescent dye design and synthesis are also providing new tools to unravel the mysteries of virus infection. With respect to new dyes, the reporting of unprecedented biophysical properties provides quantitative information about cell membranes [20], and has already been exploited in the study of HIV entry [46]. In the case of the small-sized HIV virions, exchangeable dyes constitute an interesting alternative to traditional membrane dyes, since they can circumvent photobleaching.…”

Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress

“…To connect transcriptional downregulation of glycolytic enzymes with metabolic profiling, we first used a recently developed FRET-based biosensor system in living TZM-bl cells infected with HIV-1 (San Martín et al, 2013). We combined this reporter system with labelled HIV-1 Gag to measure the stability of the glycolysis surrogate marker, lactate, at a single cell resolution using Fluorescence Lifetime Microscopy (FLIM) (Coomer et al, 2020) (Figure 2A). Data showed a significant decrease of lactate levels in cells infected with HIV-1JR-FL 3 dpi, as compared to No Env infected cells.…”