Mitch Mumby, Western University

Mitchell J Mumby1*, Cassandra R. Edgar1*, Rhiannon R.J. Woodall1, Livia G. Kucman1, Maxine E. Nicolais1, Jimmy D. Dikeakos1 1Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada *Authors contributed equally

Suppressing & Conquering

Human Immunodeficiency Virus Type 1 (HIV-1) Vpu is a small, non-enzymatic accessory protein of approximately 81 residues that enhances viral infection by modulating the surface localization of host proteins, namely tetherin, CD4, HLA-C, and Tim-3. Vpu is an integral membrane phosphoprotein composed of three alpha helices: an N-terminal transmembrane helix and two cytosolic helices connected by flexible linkers. Despite its conserved topology, Vpu sequences are highly polymorphic, with sequence variation influencing Vpu function and expression levels. While domains responsible for certain functions have been defined, the functional consequences of many naturally occurring polymorphisms remain poorly understood. Defining how Vpu polymorphisms affect protein function may provide insight into how Vpu-mediated functions are selected during infection and contribute to pathogenesis. A strategy for mapping functionally relevant polymorphisms involves generating chimeric viral proteins. We previously applied this approach to the HIV-1 Nef protein to identify determinants of CD4 and SERINC5 downregulation. However, given Vpu's small size and sequence variability, chimera generation may compromise protein stability, confounding functional interpretations. Accordingly, we developed a novel in silico workflow to evaluate the stability of lab-adapted and chimeric Vpu proteins in a simulated biological environment. Homologous Vpu structures were generated, embedded in biological membranes, and subjected to molecular dynamics simulations to assess stability. We further validated these chimeras were expressed and retained conserved Vpu functions in vitro, such as tetherin downregulation. This workflow enables downstream docking analyses to investigate the basis of Vpu functions and the impact of polymorphisms, and may inform future structure-guided inhibitor design.