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Little is known about the intermolecular dynamics and stoichiometry of the interactions of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein with its receptors and co-receptors on the host cell surface. Here we analyze time-resolved HIV-1 Env interactions with T-cell surface glycoprotein CD4 (CD4) and C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) on the surface of cells, by combining multicolor super-resolution localization microscopy (direct stochastic optical reconstruction microscopy) with fluorescence fluctuation spectroscopy imaging. Utilizing the primary isolate JR-FL and laboratory HXB2 strains, we reveal the time-resolved stoichiometry of CD4 and CCR5 or CXCR4 in the pre-fusion complex with HIV-1 Env. The HIV-1 Env pre-fusion dynamics for both R5- and X4-tropic strains consists of a three-step mechanism, which seems to differ in stoichiometry. Analyses with the monoclonal HIV-1-neutralizing antibody b12 indicate that the mechanism of inhibition differs between JR-FL and HXB2 Env. The molecular insights obtained here identify assemblies of HIV-1 Env with receptors and co-receptors as potential novel targets for inhibitor design.

Transposable elements, identified in all eukaryotes, are mobile genetic units that can change their genomic position. Transposons usually employ an excision and reintegration mechanism, by which they change position, but not copy number. In contrast, retrotransposons amplify via RNA intermediates, increasing their genomic copy number. Hence, they represent a particular threat to the structural and informational integrity of the invaded genome. The social amoeba Dictyostelium discoideum, model organism of the evolutionary Amoebozoa supergroup, features a haploid, gene-dense genome that offers limited space for damage-free transposition. Several of its contemporary retrotransposons display intrinsic integration preferences, for example by inserting next to transfer RNA genes or other retroelements. Likely, any retrotransposons that invaded the genome of the amoeba in a non-directed manner were lost during evolution, as this would result in decreased fitness of the organism. Thus, the positional preference of the Dictyostelium retroelements might represent a domestication of the selfish elements. Likewise, the reduced danger of such domesticated transposable elements led to their accumulation, and they represent about 10% of the current genome of D. discoideum. To prevent the uncontrolled spreading of retrotransposons, the amoeba employs control mechanisms including RNA interference and heterochromatization. Here, we review TRE5-A, DIRS-1 and Skipper-1, as representatives of the three retrotransposon classes in D. discoideum, which make up 5.7% of the Dictyostelium genome. We compile open questions with respect to their mobility and cellular regulation, and suggest strategies, how these questions might be addressed experimentally.

The possibility to detect and quantify protein-protein interactions with good spatial and temporal resolutions in live cells is crucial in biology. Number and brightness is a powerful approach to detect both protein aggregation/desegregation dynamics and stoichiometry in live cells. Importantly, this technique can be applied in commercial set ups: both camera based and laser scanning microscopes. It provides pixel-by-pixel information on protein oligomeric states. If performed with two colours, the technique can retrieve the stoichiometry of the reaction under study. In this review, we discuss the strengths and weaknesses of the technique, stressing which are the correct acquisition parameters for a given microscope, the main challenges in analysis, and the limitations of the technique.

HIV-1 disseminates to diverse tissues through different cell types and establishes long-lived reservoirs. The exact cellular compartment where fusion occurs differs depending on the cell type and mode of viral transmission. This implies that HIV-1 may modulate a number of common host cell factors in different cell types. In this review, we evaluate recent advances on the host cell factors that play an important role in HIV-1 entry and fusion. New insights from restriction factors inhibiting virus–cell fusion in vitro may contribute to the development of future therapeutic interventions. Collectively, novel findings underline the need for potent, host-directed therapies that disrupt the earliest stages of the virus life cycle and preclude the emergence of resistant viral variants.

Recent advances in protein labelling—gene tagging with CRIPSR-Cas9—have made it possible to label proteins of interest endogenously. This represents a major breakthrough in the field of quantitative microscopy, especially when quantifying protein-protein interactions. This is because over-expression of labelled proteins may cause a distortion in localization, function and perhaps artificially force protein-protein interactions due to crowding effects. A microscopy technique that is particularly well suited to detect protein interactions with low photon budgets is number and brightness (N&B). Detrending (removal of global trends in data) is a necessary pre-processing step to N&B calculations, but all current detrending methods perform poorly at low intensities. Here, we present the Robin Hood automatic detrending algorithm which performs well at low intensities, evaluating it with simulated and low photon budget live cell images. RH is available as an ImageJ plugin and as an R package.STATEMENT OF SIGNIFICANCEFluorescence microscopy in general and Fluorescence fluctuation methods in particular are very much dependent on detrend algorithms and so far, the user needs to decide an arbitrary number to correct for bleaching when using the box plot or the running average approach. Here, we have developed a tool available to everybody as an ImageJ plugin that is automatic, user-free and able to correct bleached images with very low photon counts.

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., Author summary Deciphering the mechanism of HIV-1 entry and fusion is crucial to understanding the first stages of infection. Although conceptually the first steps of the HIV-1 fusion reaction are relatively well understood, the role of metabolism regulating cholesterol in the plasma membrane and its impact in HIV-1 fusion has not been addressed to date. In this study, we have found a connection between host glycolytic activity and plasma membrane cholesterol concentration. These events in turn affect both membrane tension and membrane order which have a direct impact on the HIV-1 fusion reaction.