# Project 2

> **NIH NIH U54** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2024 · $1,291,540

## Abstract

THE HARC CENTER: HIV ACCESSORY AND REGULATORY COMPLEXES
PROJECT 2: REGULATION OF HIV TRANSCRIPTION AND LATENCY
SUMMARY
The HIV regulatory proteins Tat and Rev play a crucial role in controlling HIV transcription and mRNA export,
respectively. In Project 2, we will functionally and structurally elucidate the roles of ubiquitin ligases that modify
Tat, reveal the architecture of the Rev/RRE nuclear export complex to understand the mechanisms regulating
HIV mRNA translation, and compare Rev and RRE evolution in SIV and HIV to identify viral factors that contribute
to zoonosis. To achieve this, we will determine the functional interactions between the E3 ligases UBE2O and
TRAF6 with Tat by cryo-EM or X-ray crystallography (Structural Biology Core) and investigate the mechanism
by which UBE2O promotes the release of the inhibitory 7SK snRNP by ubiquitination of the HEXIM1 subunit.
We will determine the high-resolution structure of the Rev/RRE/Crm1/RanGTP nuclear export complex by cryo-
EM and define genetic and protein interaction landscapes of the Rev/RRE complex. We will also compare its
protein-protein and protein-RNA interfaces between HIV-1, HIV-2, and SIV complexes. We will use the HEPS
platform (Genetics core, Proteomics core and Structural biology core) and deep mutational scanning to
functionally validate these interactions and define them biochemically and structurally.
The latent HIV reservoir represents a significant roadblock to eradicating infection. We aim to uncover factors
that drive HIV latency, including virus integration sites and the states of chromatin and chromatin interacting
proteins. Using CRISPR-Cas9 (Genetics core), we will integrate a minimal HIV-1 LTR reporter into CD4+ T cells
to address how integration into specific genomic loci may permit expansion of particular clonal cells without virus
expression, to understand mechanistically how a large fraction of latent cells expand in chronically infected
individuals over time. Using proteomics and a variety of biochemical and biophysical methods, we will identify
post-translational modifications on HP1 proteins, test the roles of specific HP1 modifications on their phase-
separation properties, and assess site-specific HP1 mutants for virus replication. This will elucidate how the state
of chromatin and heterochromatin impact HIV transcription. Finally, we will examine the role of non-canonical
(nc) NF-kB in enhancing latency reversal for shock-and-kill therapies. We will assess synergies between
inhibition of SAMHD1 and of the ncNF-kB pathway in latency reversal in monocytic and lymphocytic latent cell
models, resting CD4+ T cells (the major reservoir of latent HIV), and gut macrophages isolated from people living
with HIV (PLWH). In summary, Project 2 will uncover potential new interfaces as HIV drug targets and evaluate
latency reversal or induction of deep latency for cure strategies.

## Key facts

- **NIH application ID:** 10829956
- **Project number:** 5U54AI170792-03
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** ALAN D FRANKEL
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $1,291,540
- **Award type:** 5
- **Project period:** 2022-07-15 → 2027-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10829956

## Citation

> US National Institutes of Health, RePORTER application 10829956, Project 2 (5U54AI170792-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10829956. Licensed CC0.

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