Fig. 2

Env-isfGFP-∆V1V2 can be packaged onto virus particles when co-expressed with wild-type Env but the particles have decreased infectivity. a Fluorescence imaging of viral particles purified from supernatants of NL4-3 and Env-isfGFP-∆V1V2 co-transfected 293T cells. Purified viruses were attached to poly-lysine treated coverslip, fixed, permeabilized and then stained by anti-p24 primary followed by Alexa Fluor 546 conjugated secondary antibody. b Two-color fluorescence plot of fluorescent HIV particles from NL4-3 or NL4-3 Env-isfGFP-∆V1V2 co-transfectanted with NL4-3. The images of purified virus particles acquired as in (a) were segmented using Imaris software and fluorescence intensity associated with Gag and Env is plotted. The gates show proportion of particles with relative sfGFP intensity > 3.2 fluorescence units. c Infectivity of supernatant viruses by Tzm-bl assay normalized by p24 ELISA. d Infectivity of Env-isfGFP-∆V1V2 when complemented with mutant HIV Env constructs. Infectivity of the same volume supernatants from indicated co-transfected cells was determined by Tzm-bl assay. e Cell-to-cell transfer of HIV Env-isfGFP-∆V1V2 requires a CD4 binding competent Env. Jurkat cells were nucleofected with indicated constructs or combination of constructs, 24 h after nucleofection, they were co-cultured with primary CD4⁺ cells, the gates show transfer of Gag to primary CD4⁺ cells. f Cell surface GFP staining of cells coexpressing Env-isfGFP-∆V1V2 and WT NL4-3. Cells were stained at 4 °C with anti-GFP antibody and AF564 secondary antibody before fixation. The red dots show sfGFP Env on cell surface. g Cell surface Env can be stained by b12, which recognizes the CD4 binding site on Env