Nef is an HIV-1 accessory protein whose function is to undermine host defenses. Long term infection with HIV strains bearing defective nef alleles leads to AIDS only over many years, suggesting Nef could be targeted as part of functional cure strategies. This basic research proposal seeks will elucidate the mechanisms of action of Nef. Nef targets include CD3, CD4, CD8, CD28, CXCR4, MHC-I, SERINC3/5, and for HIV-1 group O and SIV, BST2/tetherin. Nef substrates are downmodulated by via the clathrin-coated vesicle (CCV) pathway. Nef does not interact directly with clathrin, but rather with various members of a family of heterotetrameric adaptors known as the adaptor protein (AP) complexes, AP-1 and AP-2. The ability of the human immune system to detect and kill virally infected cells relies on proper presentation of viral antigens on the cell surface by MHC-I complexes. Nef subverts this process by promoting MHC-I complex downregulation by hijacking AP-1 and its associated small GTPase Arf1 at the TGN. MHC-I contains an incomplete version of the normal Tyr-based sorting motif. Nef complements this defective motif and converts MHC-I into a substrate for AP-1 mediated sorting to the lysosome for degradation. Structures of Nef assembled with AP-1 and the MHC-I cytosolic tail in solution suggested that Nef promotes the assembly of hexagonal lattices whose symmetry matches that of clathrin. Now, the previous solution studies will be followed up by reconstitution and structure determination of Nef, MHC-I tail, AP-1 and Arf1 in their functional setting on lipid membranes. Downmodulation of CD28 by Nefs is conserved across SIV and HIV and is mediated by AP-2. CD28 downmodulation phenotypes of Nef mutations follow a distinct pattern from other receptors, and the structural basis for this mode of CD28 is unknown. The structure of the CD28:HIV-1 Nef:AP-2 complex will be determined and leveraged to design mutations that uniquely perturb the CD28 binding site. Of the many host substrates of Nef, the most significant for viral infectivity are the multipass integral membrane proteins SERINC3 and 5. The SERINC binding site appears, on the basis of Nef phenotypes, to overlap with the site used by SIVsmm Nef to downmodulate simian tetherin, but is otherwise distinct from the known locations of CD3, CD4, and MHC-I sites. SERINCs do not share any obvious motifs with other substrates. SERINCs have been purified in monodisperse form suitable for structure determination. The cryo-EM structure of lipid- or detergent embedded SERINC3 or 5 in complex with AP-2 and HIV-1 Nef will be determined, completing a major goal in the field.

Public Health Relevance

PROJECT SUMMARY Nef is an HIV-1 accessory protein whose function is essential for the virus to undermine host defenses, yet is not targeted by any existing antirtrovirals. Nef downmodulates many substrates, including CD3, CD4, CD8, CD28, CXCR4, MHC-I, SERINC3/5, and for HIV-1 group O and SIV, BST2/tetherin by hijacking a family of heterotetrameric adaptors known as the adaptor protein (AP) complexes. This proposal will use cryo-EM and biochemistry and biophysics to determine the mechanism by which Nef hijacks AP complexes and downmodulates so many different substrates.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI120691-06A1
Application #
10092840
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Novak, Leia Kaye
Project Start
2015-06-18
Project End
2025-07-31
Budget Start
2020-08-01
Budget End
2021-07-31
Support Year
6
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Miscellaneous
Type
Organized Research Units
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94710
Morris, Kyle L; Buffalo, Cosmo Z; Stürzel, Christina M et al. (2018) HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation. Cell 174:659-671.e14
Shen, Qing-Tao; Ren, Xuefeng; Zhang, Rui et al. (2015) HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons. Science 350:aac5137