Fig. 3

Super-resolution fluorescence microscopy studies and their contribution to the understanding of HIV-1 replication cycle (illustrated in the lower panel). Virus Assembly: a dSTORM imaging of cell surface Gag distribution (green) showing representative virus-sized clusters (upper panel) and their fluorescence intensity line profiles (lower panel). Scale bar: 200 nm [105]. The density distributions of Gag protein localizations was found to be similar to the ring-like arrangement of Gag found in immature virus (see panel f). b dSTORM imaging of Env distribution (red) around cell surface Gag clusters (green). Env molecules (right panels—dots) appear to be largely excluded from the sites of Gag assembly (right panels—circle). Scale bar: 100 nm [109]. Release: c. Distribution of Gag (green) and ESCRT protein Tsg101 (red) within budding viruses imaged by dSTORM. Protein localization densities indicate the accumulation of ESCRT proteins at the neck of the virus buds [122]. d Distribution of Gag (red) and ESCRT protein Tsg101 (green) within a budding virus imaged by 3D PALM. In this study protein localization densities indicate the existence of ESCRT components within the virus particle. Scale bar: 50 nm [104]. e Tetherin clusters (red) at Gag assembly sites (green) imaged by dSTORM Scale bar: 200 nm [108]. Virus architecture and maturation: f STED imaging of Gag distribution (red) in immature and mature virus particles showing a 2D projection of ring-like Gag lattice in immature and a central condensed accumulation in mature virus particles (left panels). HIV-1 maturation kinetics was estimated by time-lapse imaging of Gag structures and quantifying the percentage of HIV-1 particles with ring-like distributions over time (right panel). Scale bar: 100 nm [100]. g STED imaging of Env distribution (red) on individual eGFP.Vpr tagged virus particles (green) with multi-clustered Env distribution in immature non-infectious particles (PR-) coalescing into a single cluster in mature fully infectious virus (wt) (right panel). Scale bar: 100 nm [112]. h sSTED-FCS measurements of Env mobility on individual mature and immature virus particles by fast line-scanning (red line) over individual eGFP.Vpr tagged virus particles (green) and determination of diffusion characteristics at each line pixel using FCS. Representative FCS correlation curve data for Env in mature (red), immature (blue) and fixed (purple) viruses with faster decay indicating increased mobility (right panel). Env was found to undergo maturation-induced increase in mobility indicating its diffusion as one of the causes for Env clustering. Scale bar: 200 nm [84]. Cell-to-cell transfer: i Visualising individual virus positions (red/yellow, identified by Gag) by STED microscopy at the contact sites between the infected macrophages (blue cell border in inset) and astrocytes (labelled via glial fibrillary acidic protein (GFAP), green) Scale bar: 500 nm. Inset scale bar: 3 µm [133]. Entry and post-entry: j. STED imaging of Env (red) and CD4 (blue) distributions in cell-attached eGFP.Vpr labelled HIV-1 (green) showing a single contact point between Env and CD4. Scale bar: 100 nm [112]. k dSTORM image of MA clusters (red) and eGFP.Vpr labelled viruses (green) after their attachment to cells. MA cluster sizes were found to be larger than those in cell-free virus particles. Scale bar: 2 µm [136]. l PALM/dSTORM image of RTC/PIC [viral DNA (red), CA (blue) and IN (green)] in the cytoplasm of infected macrophage. Scale bar: 100 nm [138]. Images were modified from indicated references with permission