Background: Microglia/brain macrophages are the principal CNS target for HIV and are infected even during apparently successful HAART, which is a threat to complete cure from HIV infection. Previously, we demonstrated that primary human adult mixed glia cultured in serum-free media can harbor virus for long periods of time (up to 77 days) with proviral integration and HIV gag protein expression, but absent robust production of infectious virus. Our hypothesis is that activation of microglia in this mixed glial culture leads to reactivation of HIV replication. We have now compared gene expression in “activated” vs “resting” cells.

Methods: Microarray analysis examined cellular RNA expression differences between cells continuously cultured in 0% or 5% FCS/GCTS for 24 hr. The data were scaled to eliminate random changes in the total amount of RNA due to experimental variability, or variation in the isolation steps in the protocol. Statistical analyses using the t-test for 2 means demonstrated significance at p < 0.05 unless otherwise noted.

Results: Microarray analysis has emphasized several major groupings of genes with increased expression in cells cultured for 24 hrs in 5% compared to 0% FCS/GCTS, despite no significant increase in total RNA or the mRNA expression in the majority of housekeeping genes. Known activation markers for microglia were significantly up-regulated. Specifically, genes in pro-inflammatory cytokine pathways (e.g., IL-1, IL-10, interferons, TNF, IFN) demonstrated significant increases in conditions of 5% FCS/GCTS, along with other activation markers such as MMPs, COX-2, and MHC class II. In contrast, known HIV transcription factors were not altered in the activated cells. Of the cellular genes (or homologs) of the ubiquination pathway that have been reported to be involved in retroviral assembly/release, only NEDD4 was significantly increased.

Conclusions: Since microglia cultured in 0% FCS/GCTS express Gag and have some HIV particles, we conclude that the block to replication in this latency model is distal to integration and transcription. Data using this microarray analysis support the notion that activation of microglia in this mixed glial cell culture correlates with robust viral replication and provide several good candidates for further investigation.