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Background:  HIV productively infects microglia and brain macrophages. Astrocytes can also be infected to a limited extent. The role of this limited, or nonproductive infection, is under investigated, although the brain is presumed to be a reservoir for HIV. Non-productive infection, characterized by early HIV gene transcripts, could be significant to neuropathogenesis. Therefore, we investigated astrocytes for viral gene transcription following HIV infection or transfection.

Methods:  Virus infection, transfection, and siRNA-mediated inhibition studies were performed. Primary human fetal astrocytes and an astrocytic cell line, SVGA, were used, along with the viral strains Ba-L, IIIB, and the infectious molecule clones YU-2 and pNL4-3YFP, the latter engineered to express yellow fluorescent protein. SVGA-LTR reporter cells were also used. Tat, Nef, and p24 were detected by immunostaining.

Results:  Following HIV (natural) infection of SVGA, very limited replication was detected using the T-tropic strains IIIB or NL4-3, while the M-tropic virus Ba-L failed to show any infection. Production of Tat and Rev persisted during the 20-day period studied, as evidenced by GFP expression. To determine if the low efficiency of infection was due to impairment or blockade of transcription, we transfected SVGA-LTR-GFP or LTR-gag-GFP cells with YU-2 or NL 4-3 proviral DNA and examined them for Tat- or Rev-mediated GFP expression. Strong fluorescence was seen 48 hours later, which was specifically inhibited by siRNA for Tat. Transfection of SVGA and primary astrocytes led to strong expression of intracellular Tat, Nef, and p24, and cell-free fluids from these cells established productive infection in Jurkat cells. While co-culture is known to be a more efficient mode of HIV transmission, co-culture of SVGA with HIV-infected Jurkat did not lead to significant enhancement of HIV infection in SVGA. Additional experiments were performed using SVGA stably transfected with HIV-GFP reporter viruses. Clones negative for p24 and Nef were selected for viral latency studies; persistence of silent HIV genomes was demonstrated by Tat-mediated induction. Co-culture of these quiescently infected cells with Jurkat resulted in infection of Jurkat.

Conclusions:  HIV infection in astrocytes is restricted at the level of viral entry. Low-level infection can occur and lead to establishment of a latent viral state susceptible to the induction of virus expression, and transmission of the infection.  

Keywords: HIV Transcription; Astrocyte infection; Latent HIV infection