Understanding the Mechanism of HIV-1 Restriction by Trim5aplha

Trim5α is a restriction factor used by mammalian cells to combat retroviral infections. This protein binds to the retroviral capsid and inhibits reverse transcription of the retrovirus. These properties of Trim5α arise from its composition, which includes an E3-Ubiquitin Ligase on its N-terminus, a B-box 2 domain, a coiled-coil domain, and a C-terminal SPRY domain. The E3-Ub ligase of Trim5α binds to the E2 protein heterodimer of Mms2-Ubc13 and long K-63 linked Ub chains are made. However, a screen of the 39 known human E2 enzymes, shows more E2 proteins can be involved with Trim5α. I hypothesize that more E2 proteins interact in Trim5α restriction.

So far, I am purifying the candidate E2 proteins. I first transform plasmids with the E2 proteins of interest into Rosetta E. Coli bacterial cells. Then I express in auto-induction media (usually ZY or LB) and lyse the cells. There is a two step purification of the lysate which involves either a nickel affinity column or an ion exchange followed by a sizing column. I will eventually perform pull down assays of the E2’s with Trim5α. Finally, I will set down crystal trays of Trim5α with cognate E2 proteins.

So far, the only result we can state is that Mms2 and Ubc13 interact with Trim5α. Since the binding experiments have not been performed as of yet, it is unclear as to what other E2’s are involved with Trim5α, but there is a high likelihood that there are more E2’s other than Mms2 and Ubc13.