FIG. 5.
Production of infectious HIV-1 is not affected in CD63 knockdown MDM with the same levels of infection. MDM were infected with HIV-1BaL 7 days after monocyte isolation (day 0) and nucleofected with scrambled or CD63-specific siRNAs 1 day after infection. Cells were returned to culture in medium containing the entry inhibitor TAK779 to avoid multiple rounds of infection. After 5 days, the medium was changed and virus-containing supernatants were collected 24 h later (i.e., at 6 dpi). (A) CD63 expression levels were assessed by Western blot analysis at 6 dpi. CHC was analyzed as a loading control. KD, CD63 knockdown MDM. (B to D) Cell-free supernatants from CD63 knockdown and control MDM were assayed for p24 levels (B) and infectivity (C), and the number of infectious units per particle were calculated (D). (E) In addition, an Alu-LTR-based real-time nested-PCR assay was used to quantify the integrated HIV DNA levels in control and CD63 knockdown MDM. Data corresponding to four experiments with three different donors were averaged and are represented as relative units after normalization with respect to control values at 6 dpi of p24, infectivity, infectious units per particle, or proviral DNA. For panels B through E, bars represent the average relative units, and error bars represent standard errors of the means.