HIV-1 Gag is translated in the cytoplasm and trafficked to the plasma membrane, where it assembles into spherical particles that bud from the cell. Proposed studies will address the structural biology, biochemistry,and molecular virology of viral membrane targeting and budding.
The first aim i s to determine the solution structure, dynamics, and membrane biochemistry of the myristoylated Gag MA domain. These studies, together with our previous structure of myristoylated MA, should reveal the molecular mechanism of the """"""""myristoyl switch"""""""" that governs Gag targeting to the plasma membrane.
The second aim i s to analyze the biochemistry of virus budding. We have identified human Tsg101 as an attractive candidate for the cellular factor that binds the Gag p6 """"""""Late"""""""" domain and facilitates virus release. Tsg101 appears to coordinate the cellular pathways of endocytosis, exocytosis, and vacuolar protein sorting (Vps), suggesting that machinery from these pathways may be recruited to assist in the viral release. We will now fully characterize the network of cellular proteins that define the different Tsg101 pathways. These studies will employ high throughput approaches for quantitating putative protein-protein interactions initially identified in automated two-hybrid screens.
The third aim i s to determine the solution structure of the N-terminal, p6 binding domain of Tsg101, both free and in complex with its p6 binding site. Our preliminary NMR and biochemical analyses indicate that this domain is structurally similar to E2 enzymes that function in the transfer of ubiquitin (denoted Tsg101 E2*) and that peptides spanning the """"""""PTAP"""""""" sequence motif of ID:V-1 p6 bind specifically to a site surrounding Tsg101 E2* beta-strand 4. The structure of Tsg101 in complex with its p6 binding site should reveal how the virus can recruit Tsg101 to assist in virus budding and may serve as the basis for structure-based design of inhibitors of viral egress.
The final aim i s to investigate the molecular virology of HIV-1 budding. We have demonstrated that dominant negative constructs of Vps4, which mislocalize Tsg101 and inhibit vacuolar protein sorting, also block HIV-1 release. We will now characterize the functional role of Tsg101 and the Vps pathway in the viral budding process.
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| McCullough, John; Clippinger, Amy K; Talledge, Nathaniel et al. (2015) Structure and membrane remodeling activity of ESCRT-III helical polymers. Science 350:1548-51 |
| Sundquist, Wesley I; Ullman, Katharine S (2015) CELL BIOLOGY. An ESCRT to seal the envelope. Science 348:1314-5 |
| Mercenne, Gaelle; Alam, Steven L; Arii, Jun et al. (2015) Angiomotin functions in HIV-1 assembly and budding. Elife 4: |
| Han, Han; Monroe, Nicole; Votteler, Jörg et al. (2015) Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs). J Biol Chem 290:13490-9 |
| Monroe, Nicole; Han, Han; Gonciarz, Malgorzata D et al. (2014) The oligomeric state of the active Vps4 AAA ATPase. J Mol Biol 426:510-25 |
| McCullough, John; Sundquist, Wesley I (2014) Putting a finger in the ring. Nat Struct Mol Biol 21:1025-7 |
| Effantin, Grégory; Dordor, Aurélien; Sandrin, Virginie et al. (2013) ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding. Cell Microbiol 15:213-26 |
| Sandrin, Virginie; Sundquist, Wesley I (2013) ESCRT requirements for EIAV budding. Retrovirology 10:104 |
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