Mechanisms of HIV accessory protein mediated control of NK cell function

Despite their potent innate cytotoxic activity, NK cells are unable to completely control HIV, however, their depletion enhances acute viremia, and their proliferation during hyperacute infection is associated with better control of infection, suggesting they do apply immune pressure. In vitro, infected cells that survive NK cell exposure have higher levels of MHC-I, suggesting that the MHC-Ilow cells are preferentially eliminated due to “missing self”. However, our preliminary data demonstrates that while genetic ablation of the HIV accessory protein, Nef, rescues MHC-I surface expression on infected cells, these cells are not completely protected from killing, suggesting mechanisms beyond “missing self” are driving NK cell stimulation. Indeed, we have discovered several new NK cell ligands that are differentially regulated with infection. Unexpectedly, we find that deletion of Vif from HIV enhances MHC-I downregulation and augments NK cell killing in co-culture assays, suggesting an additional role for Vif in controlling NK cell function. Finally, humanized mice harboring intact NK cell responses exhibit an enrichment of mutations in Nef’s acidic cluster (the dominant factor controlling MHC-I surface expression), resulting in enhanced surface expression of MHC-I. This suggests a new mechanism of viral escape from NK cell responses via changes to accessory protein regulatory elements. The overall objective of this proposal is to uncover new mechanisms of HIV accessory protein-mediated control of NK cell function. We hypothesize that while MHC-I remains the dominant regulator of NK cell activity, the virus employs additional mechanisms to fine tune this “missing self” trigger. Specific Aim #1 will identify new HIV-regulated NK cell ligands. This includes the testing of HIV clinical isolates from different clades, which exhibit accessory protein sequence diversity, and characterizing how the NK ligands CEACAM1, ICAM1, HEVM, CD43, CD44, and CD58 affect innate and ADCC-mediated NK killing. In addition, we will use mass spec to identify natural cytotoxicity receptor ligands on infected cells that trigger NK cells, and develop a custom CITE-Seq panel with an expanded repertoire of NK ligand antibodies for use in future clinical studies. Specific Aim #2 will define how HIV Vif regulates MHC-I and NK cell activation. Using mutated viruses, mRNA, and siRNA, we propose to assess how the effects of Vif on Nef may contribute to MHC-I regulation. Finally, Specific Aim #3 will characterize the effects of NK cell pressure on Nef, Vpu, Vpr, and Vif sequence evolution in vivo using humanized mice with an enhanced NK cell compartment. Mutants will be reconstructed in vitro to characterize the effects on MHC-I and other NK cell ligands, and NK cell killing. Finally, enriched mutated sequences from the animal studies will be compared to sequences in the Los Alamos HIV Database to assess their clinical relevance. Together, this work will provide mechanistic insights into viral immunoevasion mechanisms that directly affect NK cell function, which may aid the development of NK cell-based therapies for cure strategies, such as blocking accessory protein function with small molecules. Project Number: 1R01AI189316-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Kiera Clayton | Institution: UNIV OF MASSACHUSETTS MED SCH WORCESTER, WORCESTER, MA | Award Amount: $848,839 | Activity Code: R01 | Study Section: HIV Immunopathogenesis and Vaccine Development Study Section[HIVD] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI18931601A1