Alexandra Glennie, Western University

Alexandra C. Glennie (Department of Microbiology and Immunology, Western University, London, Canada), Mitchell J. Mumby (Department of Microbiology and Immunology, Western University, London, Canada), Jimmy D. Dikeakos (Department of Microbiology and Immunology, Western University, London, Canada)

Suppressing & Conquering

The HIV-1 accessory protein Nef is expressed early during infection and contributes to evasion of the host immune response. Nef modulates cellular trafficking pathways to downregulate cell surface immune effectors, including the HIV-1 entry receptor CD4. Another protein downregulated by Nef is Serine incorporator 5 (SERINC5), a multi-pass transmembrane protein that incorporates into progeny virions during egress and reduces infectivity by inhibiting fusion with target cells. Nef counteracts SERINC5 by recruiting cellular adaptor protein 2 (AP-2) to internalize SERINC5. A dileucine motif ([D/E]xxxL[L/I]165) in the Nef C-terminal region establishes interactions with adaptor proteins (APs) necessary for antagonism of SERINC5 and CD4, suggesting both proteins are downregulated through equivalent trafficking pathways. We have identified a DN164ND polymorphism within the Nef dileucine motif that decreases SERINC5 downregulation, while having minimal effects on CD4 downregulation, rendering antagonism of SERINC5 and CD4 functionally uncoupled. We hypothesize that the DN164ND polymorphism alters Nef-AP interactions during endolysosomal trafficking to uncouple these downregulatory pathways. We used the protein-protein interaction reporter assay, bimolecular fluorescence complementation (BiFC), with confocal microscopy to detect Nef and SERINC5 interactions within the endocytic pathway of transfected cells. Our analysis revealed a pronounced decrease in Rab7-positive late endosome localized BiFC associated with the DN164ND polymorphism, suggesting that this mutation compromises entry into degradative pathways. We aim to elucidate the trafficking of Nef-mediated SERINC5 downregulation by further characterizing the subcellular compartments and APs involved. This investigation may inform the development of novel therapeutics that inhibit Nef and improve outcomes in people living with HIV-1.