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. 2021 Oct 27;4:1228. doi: 10.1038/s42003-021-02658-1

Fig. 1. HIV-1 HXB2 labelling strategy and functionality of labelled viruses.

Fig. 1

a Diagram representing the DNA sequence of the plasmids used to produce HXB2 pseudotyped virions. MA: matrix. GFP: green fluorescent protein. CA: capsid. NC: nucleocapsid. Protease cleavage sites are indicated by the scissors symbol. b Schematic representation of HIV-1 immature and mature particles used in this assay. Immature particles possess the unprocessed Gag polyprotein fused to GFP. The viral protease cleaves Gag to mediate assembly of the mature HIV-1 virion. Few copies of HXB2 V4-GFPOPT embedded in the viral membrane allow analysis of Env conformations by FRET-FLIM and single-molecule approaches. c The HIV-1 Env structure representing closed Env (PBD ID 5T3X) conformation was modified to illustrate the labelling of the V4 loop with GFPOPT and NbA488 and NbA594. d Micrograph showing fluorescent HIV-1 pseudoviruses expressing Gag-GFP and HXB2 V4-GFPOPT (green) labelled with Atto 594 (red). Scale bar 10 µm. Magnification of the region delimited with a dashed contour is shown in the right column. Scale bar 2 µm. Arrows point to colocalisation events (GFP+ Atto 594+, yellow particles) which represent efficient labelling of virions. e Micrograph from transmission microscopy of TZM-bl cells infected with HIV-1 Gag-GFP viruses pseudotyped with WT HXB2 or labelled HXB2 produced in the absence (mature-enriched) or presence (immature) of the HIV-1 protease inhibitor SQV. Infected cells show a characteristic dark contrast as a result of X-gal hydrolysis upon β-galactosidase expression occurring in infected TZM-bl cells. Scale bar 0.5 mm. f Quantification of the infection efficiency (% of infected cells) in TZM-bl cells infected with equivalent amounts of HIV-1 pseudotyped viruses at different dilutions, as indicated. Dots represent the mean from three independent experiments and error bars the SD. g Quantification of the relative infection efficiency of the mature-enriched HXB2 V4-GFPOPT sample compared to the WT HXB2 pseudo-typed HIV-1 viruses. Bars represent the mean of n = 3 independent experiments, dots the individual values and error bars the SD. Unpaired two-tailed T-test (ns: p = 0.2213).