Until now it has not been possible to crystallize the intact CA hexamer. By modeling crystal structures of the individual CA domains into a 9 Å resolution EM map of assembled hexamers [2], ten possible disulfide crosslinks were designed to stabilize interactions between adjacent N-terminal domains. Engineering of the double mutants and biochemical assay identified one crosslinked hexamer as being uniquely stable and homogeneous. This stabilized hexamer yielded large single, orthorhombic crystals diffracting to 2.7 Å at SSRL beam line 7-1; the structure was solved by molecular replacement with reference to the EM model. The crystals contained two copies of CA hexamers per asymmetric unit, providing 12 independent views of previously unknown details for the interface between N- and C-terminal domains (PDB deposition 3H4E). Analysis of the structure revealed that networks of water molecules mediate interactions between hydrogen bonding residues and sites on helices of each domain, allowing the distinct variability required for capsid formation. At the same time, comparison showed that the C-terminal domain exhibits internal plasticity in the positioning of its helices (Fig. 1). The structure also afforded high resolution views of the packing of N-terminal domains around the hexamer 6-fold axis, and of the 2-fold interaction between C-terminals domain as they occur in the capsid.

The new crystal structure permits assembly of the intact HIV-1 capsid to be visualized at atomic resolution. Because the capsid performs an essential role early in the viral replication cycle, inhibition of capsid assembly by small molecules is a new therapeutic strategy for the treatment of HIV/AIDS. To date all FDA approved drugs for AIDS target the viral enzymes protease, reverse transcriptase, and integrase. The N- and C-terminal domains are attractive sites for inhibition because experimental inhibitors of HIV-1 capsid assembly target them [3, 4]. New classes of drugs that target the interface of the N- and C-terminal domains to inhibit capsid assembly, or alternatively, enhance capsid stability and prevent uncoating, would provide novel means to intervene in the virus replication cycle, and important tools to combat the evolution of resistance in HIV. The mode of binding of such compounds can now be studied in detail, and new binding sites can be identified, facilitating structure-based drug design strategies.

This study was funded by NIH grants R01-GM066087, P50-GM082545, and the George E. Hewitt Foundation for Medical Research.

Primary Citation

Pornillos, O., Ganser-Pornillos, B. K., Kelly, B. N., Hua, Y., Whitby, F. G., Stout, C. D., Sundquist, W. I., Hill, C. P., and M. Yeager, M. (2009). X-ray structures of the hexameric building block of the HIV capsid, Cell 137, 1.

References

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2. Ganser-Pornillos, B.K., Cheng, A., and Yeager, M. (2007). Structure of full-length HIV-1 CA: a model for the mature capsid lattice. Cell 131, 70-79.
3. Kelly, B.N., Kyere, S., Kinde, I., Tang, C., Howard, B.R., Robinson, H., Sundquist, W.I., Summers, M.F., and Hill, C.P. (2007). Structure of the antiviral assembly inhibitor CAP-1 complex with the HIV-1 CA protein. J Mol Biol 373, 355-366.
4. Bartonova, V., Igonet, S., Sticht, J., Glass, B., Habermann, A., Vaney, M.C., Sehr, P., Lewis, J., Rey, F.A., and Kräusslich, H.G. (2008). Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein. J Biol Chem 283, 32024-32033.