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Background:  Human astrocytes are non-productively infected by HIV-1 in vivo, infection that may contribute to the overall neuropathogenesis associated with virus infection in the brain. In vitro, infection of astrocytes with pseudotyped HIV-1 with the vesicular stomatitis envelope glycoprotein G (VSV-G/NL4-3) permits initial highly productive infection leading to a low-level virus expression, which facilitates investigation of events linked to virus replication. Mechanisms associated with regulation of viral transcription in astrocytes are poorly understood. To better understand restricted HIV-1 replication in these cells, we analyzed transcriptional regulation by chromatin immunoprecipitation (ChIP).

Methods:  Astrocytes were infected with VSV-G/NL4-3, and infection kinetics followed by p24 and by flow cytometry for expression of HIV-1 antigens. At the peak and nadir of infection, cells were collected and analyzed for the association of the transcription factors NF-kB, SP-1, and acetylated histone 4 (Ac-H4), with HIV-1 LTR.

Results:  Astrocyte infection was productive, long lasting, and reached a peak of 40% infected cells and 150 ng of viral p24 on day 7 post-infection. In contrast, at the lowest point at day 30 post-infection, 24% of the cells expressed viral antigen and 44 ng extracellular p24. ChIP analysis at the 2 time points post-infection showed no difference in the levels of NF-kB binding to the HIV-1 LTR, and 4-fold decrease in SP-1 binding on day 30. The levels of NF-kB and SP-1 binding to the HIV LTR on day 7 post-infection appeared to be equal. Ac-H4 levels were 1.7-fold lower on day 30 post-infection than on day 7.

Conclusions:  These results demonstrate that in vitro, primary human astrocytes are productively infected by HIV-1, leading to a gradual decline and basal low-level viral replication. The fact that levels of NF-kB did not change considerably throughout infection indicates continuous involvement of NF-kB in HIV-1 transcription. In contrast, a 4-fold decrease in SP-1 on day 30 post-infection suggests an active role for SP-1 in down-modulation of viral transcription. A 1.7-fold decrease in Ac-H4 at the nadir of infection confirms the overall decrease in transcriptional activity, as well as implies a possible nucleosome rearrangement on the LTR promoter. The overall decline in transcription correlates well with a decrease in p24 levels and percentage of positive cells on day 30 post-infection.

Keywords: HIV-1; Transcription; Human astrocytes