Characterizing the minimal domain of eIF3f responsible for HIV-1 inhibition

Human Immunodeficiency Virus-1 (HIV-1) is an epidemically established retrovirus infecting more than 30 million individuals worldwide. HIV infects human host CD4+T cells that are vital to the innate immune system, resulting in severe immunodepression. Valente (et. al. 2006 and 2009) have identified the N-terminal portion of the eukaryotic initiation factor 3 subunit f (N91-eIF3f) as a potent inhibitor of HIV-1 replication. Overexpression of N91-eIF3f and full-length protein inhibit production of mature transcribed viral mRNA by blocking the cleavage step during HIV-1 3’ end RNA processing. To identify the minimal domain of eIF3f necessary for the inhibition of HIV-1 we used Polymerase Chain Reaction for cloning the expression vector several eIF3f fragments spanning the entire protein. Vectors expressing the eIF3f fragments were transfected into Hela-CD4+ cells and stable cell lines were established by positive hygromycin drug selection. These cells will then be infected with wild type (wt) HIV-1. To identify the specific fragment of eIF3f responsible for HIV-1 3’ end processing inhibition we will quantify by p24 ELISA amounts of capsid released into the media. Fragments that would restrict the virus, as does N91-eIF3f, will induce a reduction of amount of capsid produced. A side project was to determine if endogenous eIF3f was necessary for N91-eIF3f inhibition of HIV-1 (wt) and HIV-2. We made stable Hela-CD4 cell line with N91-eIF3f knock-downed for endogenous eIF3f. Infection of these stable cell lines with HIV wt will be performed to confirm the importance of endogenous eIF3f in the inhibition mediated by N91-eIF3f.