# Project 1

> **NIH NIH U54** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2024 · $896,937

## Abstract

THE HARC CENTER: HIV ACCESSORY AND REGULATORY COMPLEXES
PROJECT 1: STRUCTURE AND EVOLUTION OF THE VIF-APOBEC3 COMPLEX
SUMMARY
In Project 1 we will elucidate novel structural aspects of the APOBEC3 (A3) family of restriction factors and how
they are antagonized by the HIV accessory protein Vif. Primate Vif targets A3’s for degradation by the 26S
proteasome, but it is unknown how Vif intercepts A3 packaging complexes. It has been suggested that Vif binds
different A3 family members through three different interfaces, but whether these binding sites are independent
or dependent on one another is unclear. In previous studies, we uncovered that Vif forms functional interactions
with additional host factors, including regulatory subunits of PP2A, components of the chromatin-modifying and
transcriptional machinery, and regulators of ubiquitin-mediated proteolysis. We will now investigate how Vif
neutralizes different A3 family members to promote efficient viral replication, and how adaptations in Vif enabled
neutralization of A3G in hominid primates and how these adaptations affect the ability of human A3G to escape
HIV-1.
We will determine the structure of A3G and PP2A regulatory subunits bound to Vif using cryo-EM (Structural
Biology Core), as well as deep mutational scanning (DMS) (Genetics Core), to uncover the mechanisms by
which Vif recognizes different substrates and multitasks the degradation of A3 and PP2A subunits. The functional
significance of structural observations will be further tested using viral and biochemical assays, and DMS in
primary CD4+ T cells will explore tradeoffs between the ability of Vif to neutralize specific A3 family members vs.
others. We will also use cryo-EM (Structural Biology Core), functional studies, and DMS (Genetics Core) to
determine how Vif’s ability to engage restriction factors is rewired by adaptations allowing cross-species
transmission. This is important because A3G and Vif undergo repeated bouts of positive selection and adaptation
in what has been termed a ‘molecular arms race’, a process which led to cross-species transmission and the
birth of HIV-1. Finally, we will investigate the mechanism of A3 packaging in the absence of Vif by determining
composition and architecture of A3 packaging complexes, a long-standing question in the field. We will use the
HEPS platform to discover host and viral proteins required for packaging of newly synthesized A3 family
members (Proteomics Core). CRISPR-Cas9 and mutagenesis will determine the functional significance of the
A3 packaging complex (Genetics Core). Cryo-EM studies will be performed on the packaging complex
(Structural Biology Core). These approaches will provide snapshots of A3 family members en route to
packaging and define how Vif intercepts these structures to promote viral infectivity. Discoveries made by
Project 1 will enable rational drug design to target HIV-1 from establishing replication-competent proviruses by
utilizing the restriction potential...

## Key facts

- **NIH application ID:** 10829954
- **Project number:** 5U54AI170792-03
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** John D Gross
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $896,937
- **Award type:** 5
- **Project period:** 2022-07-15 → 2027-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10829954

## Citation

> US National Institutes of Health, RePORTER application 10829954, Project 1 (5U54AI170792-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10829954. Licensed CC0.

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