| Description |
The Gag protein of human immunodefleiency virus (HIV) contains a series of domains and motifs that drive virus assembly, package components of the virus, and recruit the cellular machinery required for formation of infectious virus particles. The C-terminal domain of Gag, termed p6, contains two short motifs that perform recruitment functions. A P(T/S)AP tetrapeptide motif binds the cellular protein TsglOl (tumor susceptibility gene 101) and is required for the release of new virus particles from the surface of infected cells, whereas an LXXLF motif binds the HIV-1 protein Vpr (viral protein R) and ensures its packaging into the assembling virion. To understand how p6 performs these recruitment functions, p6-Tsgl01 and p6-Vpr complexes were generated in vitro and characterized biochemically and structurally. The N-terminal UEV domain of TsglOl, a ubiquitin E2 enzyme variant, binds directly to the p6 PTAP motif (Kd = 27±5 ^lM). NMR structural studies of TsglOl UEV, both free and in complex with a PTAP peptide derived from HIV-1 p6, revealed that it is structurally similar to E2 ubiquitin Ligase enzymes, but contains an extra N-terminal helix, an extended p-hairpin, and lacks two C-terminal heHces. The PTAP peptide occupies a hydrophobic groove on the TsglOl UEV surface that is exposed by the absence of the C-terminal helices. The two PTAP prolines are bound by two pockets along the binding groove: one that recognizes the N-terminal Pro, and a second that recognizes the C-terminal Ala-Pro dipeptide. Interestingly, the Ala-Pro pocket uses the same mechanism of proline recognition as X-Pro pockets of SH3 and WW domains. Mutations in either PTAP or TsglOl UEV that reduce binding also reduce virus particle release. TsglOl is therefore an attractive target for the development of novel drugs against HIV. TsglOl UEV also binds the protein ubiquitin, albeit weakly (Kd = 635±82 ^M), using the large concave surface of the p-sheet. Ubiquitin enhanced the affinity of p6 by 10-fold, consistent with a role for ubiquitin in modulating TsglOl UEV interactions. Biochemical studies revealed that HIV-l p6 binds Vpr and a construct spanning the first 71 amino acids (Vpr 1-71) with similar affinity (Kd = -75 ^iM). Mutations in both Vpr and the p6 LXXLF motif that reduce Vpr packaging also reduce binding in vitro, and there is therefore good correlation between the biochemical and genetic data. Vpri-71 forms trimers and aggregates in solution, suggesting that self-association may facilitate Vpr virion incorporation by increasing its avidity to assembling Gag molecules at the plasma membrane. In summary, this thesis provides a biochemical and structural basis for understanding how the p6 domain of the HIV-l Gag protein recruits other proteins that function in viral replication and budding. |
