Figure S1.

Characterization of purified NNHIV particles and the effect of IN.mScarlet incorporation on HIV-1 infectivity, related to Figure 1

(A, B) Immunoblot analysis of virus particles purified from the supernatant HEK293T cells transfected with proviral plasmid pNLC4-3 (A, lane 1), pNNHIV (A, lane 2), pNNHIV and pVpr.IN_D64N/D116N.eGFP (B, lanes 1) or pNNHIV and pVpr.IN_D64N/D116N.mScarlet (B, lanes 2). Antisera raised against recombinant HIV-1 CA, RT or IN, or against eGFP was used. (A) Wild-type (WT) HIV-1 virions and NNHIV particles have a similar composition and Gag processing. (B) NNHIV particles labeled with IN fused to eGFP or mScarlet fluorescent protein (FP) have a comparable composition, Gag processing and FP-fused IN incorporation. (C, D) Effect of IN.mScarlet incorporation on HIV-1 infectivity. Particles were purified from the supernatant of HEK293T cells transfected with plasmid pNLC4-3 (HIV-1) or pNL4-3 and pVpr.IN.mScarlet (HIV-1 IN.mScarlet). (C) Analysis of labeled virus particles by immunofluorescence. Virions were adhered to PEI-coated 8-well chamber glass bottom, fixed and immune-stained using antiserum against HIV-1 CA. Representative confocal images recorded in the CA (green) and IN.mScarlet (red) channel. The presented fraction (%) of labeled virions (n = 1,036) was quantified using spot detector of the Icy software as described in Materials and Methods. (D) Infectivity of IN.mScarlet-labeled virions. SupT1-R5 cells were infected with equal amounts of non-labeled or IN.mScarlet-labeled HIV-1_NL4-3 virions for 24 h prior to addition of T-20 fusion inhibitor. At 48 h p.i., cells were fixed and immunostained for intracellular HIV-1 CA. Levels of infected cells were scored by flow cytometry. The graph shows mean values and SEM from three independent experiments performed in triplicates.