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3D Electron Microscopy Of Molecular Assemblies
Zhang Lab University of Pittsburgh School of Medicine Department of Structural Biology
 
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Mechanisms of HIV-1 capsid assembly, maturation and disassembly

Retroviruses, such as human immunodeficiency virus 1 (HIV-1), contain mature conical capsids that enclose the viral RNA genome, enzymes and accessory proteins. The assembly and stability of the viral capsid are critical to the viral replication life cycle. Retroviral capsid is comprised of capsid protein, which contains two structural domains that are connected by a flexible linker. Structures of individual domains and the full-length protein, as well as hexamic and pentameric CA were determined to atomic level, providing a comprehensive gallery of assembly subunit of HIV-1 capsid. However, the mechanisms of capsid formation, which requires not only the molecular structures of the building blocks, but also the inter-subunit interactions necessary for the assembly of an asymmetric capsid, remain unclear. Because the structure of the capsid lattice has important implications for our understanding of capsid assembly and disassembly and for the development of therapeutic drugs that target viral capsid, reliable high resolution structural models of assembled HIV-CA and maturation intermediates are necessary. We are using cryoEM methods, combined with large scale all-atoms molecular dynamic simulation and biochemical validation to provide reliable structural models of capsid assembly.

 

Zhao G, Perilla J. R., Yufenyuy E. L., Meng X., Chen B., Ning J., Ahn J., Gronenborn A. M., Schulten K., Aiken C. and Zhang P.* (2013). Structure of the Mature HIV-1 Capsid by Cryo-EM and All-Atom Molecular Dynamics Simulation. Nature 497, 643-646. Featured on Nature Cover.

Meng X., Zhao G., Yufenyuy E., Ke D., Ning J., DeLucia M., Ahn J., Gronenborn A.M., Aiken C.* and Zhang P.* (2012) Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid. PLoS Pathog 8(8): e1002886.

Byeon In-ja L., Meng X, Jung J., G. Zhao, Yang R., Shi J., Ahn J., Concel J., Aiken C., Zhang P.* and Gronenborn A. M.* (2009) Structural Convergence between CryoEM and NMR Reveals Novel Intersubunit Interactions Critical for HIV-1 Capsid Assembly and Function. Cell 139: 780–790.

HIV-1 and host cell interactions

Following fusion of the viral and host membranes, the HIV-1 core is released to the cytoplasm of the host cell. The viral capsid disassembles in a process termed uncoating and reverse transcription of viral genome takes place before transporting into cell nucleus for integration. Many cellular factors inference viral processes within the host cell, in particular those interacting with viral capsid, including host dependency factor, such as CypA, and restriction factors, such TRIM5α or TRIMCyp. The peptidylprolyl isomerase CypA binds to the outer surface of assembled CA, facilitating infection by an unknown mechanism. To define the structural and functional effects of CypA on capsid uncoating, we are carrying out structural studies of HIV-CA/CypA complexes using cyroEM methods, combined with mutagenesis and functional assays.

Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. TRIM5α is a tripartite motif protein, with RING, B-box, coiled-coil (CC) and C-terminal B30.2/SPRY domain. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. Using cryoEM, combined with mutagenesis and chemical cross-linking, we are investigating the direct interactions between HIV-1 CA assemblies and the host restriction factor TRIM5αrh. Extending our studies between a recombinant fragment of the restriction factor TRIM5α, consisting of the CC and SPRY domains of rhesus macaque TRIM5α, and tubular assemblies of HIV-1 CA protein that revealed how direct structural damage is imparted on CA tubes, we aim to assess the structural influences of the B box and RING domains in TRIM5α’s known ability to block HIV-1 reverse transcription.

Liu C., Perilla J. R., Ning J., Lu M., Hou G., Ramalho R., Bedwell G., Byeon I., Ahn J., Gronenborn A. M., Prevelige P., Rousso I., Aiken, C., Polenova T., Schulten K. and Zhang P.* (2016) Cyclophilin A Stabilizes the HIV-1 Capsid through a Novel Non-canonical Binding Site. Nat Commun. 7, 10714.

Yang H., Ji X., Zhao G., Ning J., Zhao Q., Aiken C., Gronenborn A. M., Zhang P. and Xiong Y. (2012) Structural Insight into HIV-1 Capsid Recognition by Rhesus TRIM5α. Proc Natl Acad Sci  USA 109(45): 18372-1837.

Zhao G., Ke D., Vu T., Ahn J., Shah V. B., Yang R., Aiken C., Charlton L. M., Gronenborn A. M. and Zhang P.* (2011) Rhesus TRIM5α Disrupts the HIV-1 Capsid at the InterHexamer Interfaces.  PLoS Pathog 7(3):e1002009.

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