Presentation description

In order for HIV to exit infected cells, it hijacks the endosomal sorting complexes required for transport (ESCRT) pathway, which normally mediates cellular membrane fission events in healthy cells. A truncated version of the ESCRT-III factor CHMP3, known as retroCHMP3 has been found in New World monkeys and mice RetroCHMP3 is able to block this ESCRT-dependent virus budding without interfering with the ESCRT pathway's important cellular functions. Consequently, those cells are able to inhibit HIV replication while remaining healthy. In order to further examine the effects of retroCHMP3 on HIV replication, we used CHMP3 constructs, produced by the Nels Elde lab, that are able to produce the truncated retroCHMP3 when cleaved by either TEV protease or HIV protease. We are attempting to transfect 293T cells with these constructs and the appropriate protease, and then adding envelope-free HIV. By comparing the concentrations of HIV between our control treatment and the protease treatments, we will be able to discern how effective this cleavage method is in cleaving the constructs, thereby expressing retroCHMP3 and reducing HIV replication. We are using a p24 ELISA to determine if virus is present, which would tell us if replication was inhibited by retroCHMP3. We are also performing western blots with retroCHMP3 and HA antibodies to determine if cleavage into retroCHMP3 has occurred.