Figure 2.

PICALM KO increases expression and colocalization of virus and autophagy proteins, can produces more virus, but have diminished infection rates

(A and B) KO cell lines were infected with WT NL4-3 provirus (MOI = 5; n = 3) for 48 h. Cells were treated with 0.1% saponin to deplete LC3B-I from cells. Cells were collected for FISH analysis for the HIV-1 vRNA and immunofluorescence for HIV-1 Gag protein and host proteins LC3B-II and LAMP1.

(C) Corresponding boxplots demonstrate that PACSIN3 KO caused increased LAMP1 and its colocalization with LC3B-II. LC3B-II colocalization with the vRNA is also increased in PACSIN3 KO. PICALM KO caused increased LC3B-II expression, increased Gag expression, along with increased LC3B-II colocalization with Gag. VAMP2 KO caused increased LAMP1, and increased colocalization between LC3-II and Gag, LC3B-II and the vRNA, and Gag and the vRNA. Note: LAMP1ՈLC3B-II colocalization in the PICALM KO is particularly low and is thus practically juxtaposing the x axis (2.33 ± SD 2.06 MFI).

(D) Lysates were collected and immunoblotted for Gag p55 and p24.

(E) Bar graph from data in (D).

In parallel, culture supernatant was collected and (F) virus were determined by ELISA p24. One-Way ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Error bars from bar graphs indicate ±SD. MFI, mean fluorescence intensity; %, percent; rel., relative; UI, uninfected; Inf., infected. Ո, colocalization (i.e., intersection); KO, knock out; μm, micrometer.