The Role of FEZ Protein Homologs in Early HIV-1 Infection

Northwestern University At Chicago, Evanston IL

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT A critical yet still poorly understood step in HIV-1 infection is the early post-entry trafficking of viral cores across the cytoplasm to reach the nucleus. This requires directed and controlled transport along microtubule filaments by host motor complexes, but precisely how viral cores control these processes is only beginning to be defined. This is in part due to the unusual structure of the HIV-1 core, which is a metastable fullerene cone composed of ∼250 hexamers and 12 pentamers of capsid protein (CA), and its equally unusual and enigmatic strategies for engaging microtubule motors. Our lab was the first to demonstrate that incoming HIV-1 particles induce the formation of specialized stable microtubule subsets to facilitate their long-range transport to the nucleus. We subsequently discovered the first HIV-1-associated cellular motor adaptor, Fasiculation and Elongation Factor Zeta 1 (FEZ1). Binding to FEZ1 enables viral recruitment and control over the outward- directed motor, kinesin-1, which often exhibits higher affinities for stable microtubules. Using a variety of genetic and biochemical approaches combined with live cell imaging of infection in biologically relevant cell types, our data further revealed that localized phosphorylation of FEZ1 on viral cores allows HIV-1 to regulate kinesin-1 activity. This in turn controls the bi-directional movement of virus particles, a key process that allows cargos to navigate the densely crowded cytosol while ultimately favoring net forward displacement to reach the nucleus. While FEZ1 phosphorylation enables viral control over Kinesin-1, our data now demonstrates that distinct coiled coil (CC) domains in FEZ1 mediate its binding to viral capsids through high avidity charge-based recognition of hexamer pores, yet the exact structural basis of these interactions and their functional roles in infection remain unknown. Preliminary data further shows that each of FEZ1’s four CC domains play a role in infection. Based on structure modeling using AlphaFold3, we hypothesize that CC2 and CC4 mediate binding to capsids while CC1 and CC3 regulate kinesin-1 engagement. Additional preliminary data further reveals that FEZ2 is a structurally and functionally divergent paralog of FEZ1. Structural comparisons suggest that FEZ2 contains distinct CC domain organizations that still mediate interactions with HIV-1 capsids but lacks kinesin- regulatory domains. In line with this, we find that FEZ2 inhibits HIV-1 trafficking to the nucleus and the establishment of infection. Furthermore, our data demonstrate that FEZ2 is differentially expressed relative to FEZ1 in different biologically relevant cell types. From these data, we hypothesize that divergence in their CC domains underlies the distinct pro- and anti-viral activities of FEZ1 and FEZ2 proteins, respectively, and their relative expression levels serve as an important determinant of cellular susceptibility to infection. Developing a detailed structural and functional understanding of how FEZ family members interplay and exert opposing effects on infection will not only provide fundamental insights into poorly understood aspects of HIV-1 infection but may also guide the future development of targeted antivirals that disrupt these capsid-based interactions.