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bioRxiv · 10.64898/2026.04.15.718738

Quantitative and mutational analysis of soluble HIV-1 Vpu and calmodulin interactions

Ogunbowale, A.·Hadadianpour, E.·Ishola, O.·Islam, M. M.·Ramos, N.·Saffarian Delkhosh, A.·Georgieva, E. R.
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Abstract

HIV-1 Vpu supports viral adaptation through host-protein interactions. Although mainly membrane-associated, we recently identified a soluble Vpu form that forms a stable complex with Ca2+-bound calmodulin (Ca2+-CaM), potentially influencing Vpu trafficking. Here, to determine the binding affinity and identify regions of soluble Vpu involved in CaM binding, we used ensemble Forster Resonance Energy Transfer (eFRET). We tested Cy3-labeled full-length (FL) Vpu, a C-terminal fragment (helices 2 and 3), and a Cy3-labeled FL Vpu V22A/W23Y/I33N mutant with substitutions of key residues in Vpu helix 1 and helices 1-to-2 loop having a role in the interaction with Ca2+-CaM. All Vpu variants were labeled at residue L42C. Ca2+-CaM was tagged with Cy5 at residue S39C. eFRET analysis of 100 nM Cy3-Vpu variants mixed with Cy5-Ca2+-CaM (in the range 100 nM-2.5 microM) revealed heterocomplexes formation with dissociation constants (Kd) and binding free energies (deltaG). FL Vpu-Ca2+-CaM showed highest stability (Kd ~74 nM, deltaG ~-9.7 kcal/mol), while the truncated C-terminal region and V22A/W23Y/I33N mutant formed weaker complexes with Ca2+-CaM (Kd ~182 nM and 800 nM, deltaG ~-9.2 kcal/mol and ~-8.3 kcal/mol). The reduced Vpu-Ca2+-CaM stability after disruption of binding sites in and near Vpu helix 1 may explain how this interaction is regulated, including through lipid competition that promotes Vpu membrane insertion. We propose that, at the membrane, hydrophobic helix 1 dissociates from Ca2+-CaM and inserts into the lipid bilayer, weakening the complex and releasing CaM. These findings clarify HIV-1 Vpu interactions with cellular components and may inform antiviral development.

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Ogunbowale, A., Hadadianpour, E., Ishola, O., Islam, M. M., Ramos, N., Saffarian Delkhosh, A., Georgieva, E. R.. 2026-04-16. Quantitative and mutational analysis of soluble HIV-1 Vpu and calmodulin interactions. https://doi.org/10.64898/2026.04.15.718738

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