Figure 3:

Long term HIV infection of astrocytes is reactivable. (A) Long term infection time course, up to 120-150 days in the presence of HIV_ADA, HIV_BaL, HIV_JRCSF, HIV_JRFL, and HIV_98UG021. The time course in Fig. 2B was not viral strain-specific and laboratory-adapted, and primary isolates behave similarly after HIV-infection of primary cultures of astrocytes. (B) Viral reactivation (React, see arrows) after 120 days in culture with SAHA (1 μM), TNF-α (10 ng/mL), or Methamphetamine (1 μM). Upon addition of the reactivating agent, HIV-p24 in the supernatant was quantified by ELISA up to 296 days post-infection (*p≤0.0024 as compared to uninfected conditions, in Graph B, all curves were significant as compared to HIV infection alone except 216, 240, and 296 days post-infection with a p≤0.004). (C to J) correspond to a representative example of staining for Uninfected astrocytes (C-F) and HIV infected astrocytes (G-J). Denote the colocalization of the HIV-Nef DNA signal with DAPI and Alu-repeats. Bar: 15 μm. (K) Quantification of the total number of astrocytes with HIV integrated DNA (HIV-DNA) expressing viral mRNA (HIV mRNA) and HIV-p24 (HIV-p24) during long-term culture. (L) Quantification of the numbers of HIV-infected astrocytes after reactivation with SAHA/TNF-α/methamphetamine. All numbers were pooled together due to the low variability. At each time point, three coverslips were fixed and stained for HIV-DNA, mRNA, and p24. Denote the increase in viral mRNA and protein expression n=5-7 independent experiments from different donors with three coverslips per time point.