Structural studies of the HIV-1 CA protein

• Main Author: Kelly, Brian N
• Format: Dissertation
• Language: English
• Subjects: biophysics, medical; chemistry, biochemistry
• ISBN: 9780549428619; 0549428615

The core of HIV-1 is enclosed in a capsid, whose assembly is a vital step late in the viral life-cycle. The capsid is comprised of the two-domain CA protein, whose amino-terminal domain (NTD) forms hexamers and whose carboxy-terminal domain (CTD) is a stable dimer in solution that mediates dimeric interactions between hexamers within the capsid. CA is expressed as the largest segment of the Gag polyprotein, which is processed by the viral protease during viral maturation to yield three main structural proteins, MA, CA, and NC, and three smaller polypeptides, SP1, SP2, and p6. Gag processing and the transition from an immature to a mature particle involves local structural changes, such as formation of the CANTD beta-hairpin, that are coupled to larger global rearrangements such as formation of the capsid.
To understand viral maturation better, I determined a crystal structure of MA-CANTD, which is the first 278 residues of Gag and a new structure of CANTD. These structures challenged earlier proposals that MA-CA processing induced mechanistically important conformational changes in CANTD, and indicated that processing-induced changes to the CANTD conformation are modest and almost entirely limited to formation of the CA N-terminal beta-hairpin. These observations support the model that the human protein cyclophilin A functions in the viral life cycle by modulating interactions with host immunity factors rather than by directly promoting capsid disassembly.
In an effort to develop new therapeutics that target viral capsid assembly, I crystallized CANTD in the presence of the weak binding assembly inhibitor CAP-1 and determined the structure. Remarkably, CAP-1 induces displacement of the buried Phe32 side chain to a highly solvent exposed environment, although the CAP-1 molecule itself was not resolved. I therefore collaborated with Michael Summers to perform a joint X-ray/NMR refinement and determine details of the interaction. Analysis of multiple structures indicates that the Phe32 transition relieves conformational strain, thereby suggesting that this transition may perform a normal role during HIV replication. Superposition of our CA NTD:CAP-1 structure with a recent low resolution reconstruction of the capsid lattice indicates that this class of inhibitors functions to destabilize the CA hexamer by blocking an NTD-CTD interaction.