Molecular Basis of MHC-I Downregulation by HIV-1 Nef via AP1

Rajendra Singh, Veterans Medical Research Foundation, San Diego
Mentor: John Guatelli
Training in Basic Biomedical Sciences
Postdoctoral Fellowship Award

Human immunodeficiency virus type 1 Nef is a 27kDa, myristolated protein with no enzymatic functions. Nef has an important role in the pathogenesis of HIV-1 and SIV viruses. Downregulation of the major histocompatibility complex class I (MHC-I) from the surface of infected cells by Nef likely contributes to pathogenesis by providing evasion of cell-mediated immunity. Nef-induced downregulation of MHC-I involves endosomal trafficking and the clathrin adaptor protein complex-1 (AP-1). MHC-I, Nef and AP-1 participate in a cooperative interaction. The basis of this interaction is poorly understood: the minimal components are not defined, and its structural basis is unknown. The long-term objective of this proposal is to solve the crystal structure of the complex formed between MHC-I, Nef, and AP-1 and to establish the nature of this host-pathogen protein-protein interaction at the molecular level. To achieve this objective we have three specific aims: 1) express the protein components in large scale; 2) validate that the proteins are sufficient to constitute a biologically relevant interaction; and 3) solve the crystal structure of the complex. These specific aims would be achieved by the following experimental approaches: 1) cloning, expression and purification of the MHC-I cytoplasmic domain, Nef, and the ì1 subunit and/or the AP-1 complex core: 2) identification and characterization of the protein components necessary for a biologically relevant complex; and 3) crystallization of the MHC-I/Nef/AP-1 complex and determination of the structure of the complex by X-ray crystallography. This research would identify the key binding sites within the MHC-I CD/Nef/AP-1 complex. Novel interacting interfaces between the protein components are likely to be discovered. This information could enable a structure-based rationale for the design of new antiviral drugs for the treatment of HIV-1 infection. HIV-1 infection and AIDS remains an important medical problem in California and the world. A key problem is the persistence of HIV-1 in the infected person. Drugs that target HIV-1 Nef could help address this problem, by allowing more effective immune surveillance and clearance of infected cells. Our proposed effort to elucidate the precise molecular basis of the downregulation of MHC-I by Nef could be an important step toward realizing Nef as a novel target for therapy.