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Background: We have previously reported a model for low-level, long-lived infection of microglia/brain macrophages under non-activating conditions (no serum or cytokines).  We determined that these cells could be infected with little to no viral output compared to the productive infection of activated microglia; whereas activation resulted in robust viral output.  Molecular analysis of the viral life cycle in these cultures led us to focus on the later events, namely assembly and budding.  In order to identify proteins that interact with the HIV Gag protein within microglia or macrophages we performed a number of co-immunoprecipitation experiments, comparing proteins that associate with Gag in activated and non-activated cultures.

Methods:  A combination of immunoprecipitation of the Gag protein from HIVYU-2 infected microglia with mass spectometry analysis was used to identify potential host protein candidates for our model.  Follow up analysis employed immunoprecipitation of purified tagged proteins and subsequent western blotting for suspected binding partners.

Results:  We have identified several cellular proteins with interact with HIV-1 Gag in macrophages and microglia.  Of specific interest were proteins involved in the endosomal sorting pathway and exocytosis, processes that have recently been shown to be critical to HIV replication in macrophages. One of these candidate proteins, which will be discussed in the meeting, appears to have macrophage specific expression when compared to peripheral blood lymphocytes. 

Conclusions:  Our analysis suggests that this approach may aid in identifying further cellular partners that participate in HIV assembly and budding.  Furthermore, while many of these proteins may play a role in HIV replication in both T cells and macrophages, this method is useful for identifying macrophage specific proteins important to HIV replication.  These interactions could aid in understanding HIV budding into and release from the intracellular multi-vesicular body network of macrophages, as well as provide new therapeutic targets.

Keywords: macrophages/microlgia; budding/assembly; Gag