# The Multiple Functions of Vpu at the Membrane

> **NIH NIH P50** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2020 · $35,068

## Abstract

HIV Viral Protein U (Vpu) is an essential, transmembrane viral protein with several functions in modulating the
host cellular environment for optimal viral replication. First, Vpu plays a critical role in remodeling the cell
surface by removing membrane-bound host proteins that inhibit viral replication, including CD4, BST-2/Tetherin,
and MHC molecules. It does this by recruitment of the Cul1-βTrCP-Skp1-Rbx1 E3 ligase complex to the
membrane, which subsequently ubiquitinates and removes target proteins from the cell surface by retrafficking
and/or degradation. Second, Vpu is known to influence immune signaling, particularly through deregulation of
NFKb. This occurs through both sequestration of βTrCP, which would otherwise activate NFKb by degradation
of its IKK inhibitor, and through down-regulation of BST-2, which is thought to activate NFKb through an
interaction between its cytoplasmic domain and TRAF. Third and finally, Vpu is thought to act as a homooligomeric
viroporin ion channel in the Golgi apparatus to alter membrane potential and potentially enhance
virion release. All three functions rely on the coordination of multiple events at the cell membrane in
conjunction with a series of characterized and yet unknown host protein complexes. An imperfect
understanding of the host complexes involved and the inherent difficulties of working with membrane proteins
in vitro has stifled our ability to understand how Vpu carries out each of these distinct processes. To better
characterize the multifunctional nature of Vpu, we propose to employ an overall strategy that couples state-of-the
art proteomic discovery with structural/biophysical mechanistic interrogation and primary cell genetic
validation. In Aim 1, we will employ global proteomic techniques including post-translational modification (PTM)
profiling and Ascorbate Peroxidase-based proximity biotin labeling mass spectrometry (APEX-MS) to identify
the host complexes and signaling pathways engaged by Vpu and select separation-of-function mutants (Core
1 and 5). In Aim 2, we will employ a combination of high-throughput mutagenesis and antibody-derived binding
partner stabilization approaches to obtain cryo-EM and X-ray diffraction structures of monomeric and homooligomeric
Vpu complexes at atomic resolution (Cores 3, 4, 6 and 7). Candidate host binding factors identified
in Aim 1 will be tested for Vpu binding in vitro by Fluorescence Size Exclusion Chromatography (FSEC) and
similarly used for structural interrogation. In Aim 3, we will employ primary cell CRISPR/Cas9 editing
approaches to knock-out each of the candidate host factors identified in Aim 1 to test their impact on the
replication of a series of vpu mutant viruses (Core 2). Protein-protein interactions and localization in the
presence and absence of Vpu will be validated in vivo by confocal microscopy. Working with Core 5, our data
will be collated for structure-function hypothesis generation that we will ultimately test in our pri...

## Key facts

- **NIH application ID:** 9993243
- **Project number:** 5P50AI150476-14
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Robert M Stroud
- **Activity code:** P50 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $35,068
- **Award type:** 5
- **Project period:** 2007-08-27 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9993243

## Citation

> US National Institutes of Health, RePORTER application 9993243, The Multiple Functions of Vpu at the Membrane (5P50AI150476-14). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9993243. Licensed CC0.

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