# Structural interrogation of the HIV-1 5′ leader RNA by multidimensional chemical mapping and cryoelectron microscopy

> **NIH NIH R21** · STANFORD UNIVERSITY · 2020 · $195,625

## Abstract

PROJECT SUMMARY
The 5′ untranslated region (5′ leader) of the HIV-1 RNA genome is critical for HIV viral replication. The presence
of numerous highly conserved structural elements in this region raises the prospect of novel anti-HIV
therapeutics that could bind or disrupt the secondary structure, tertiary folding, and RNA-protein interactions of
these elements. Yet, despite decades of investigation of the 5′ leader RNA, there remains substantial controversy
over the 5′ leader’s global structure, hindering our basic understanding of the HIV life cycle and delaying our
ability to develop curative therapeutics. Previous studies using low-information structural techniques have led to
nearly twenty conflicting single-state secondary structure models and numerous long-range tertiary contacts that
have been difficult to confirm or refute; indeed, it is possible that the RNA forms numerous distinct biologically
important structures. We have recently invented multidimensional chemical mapping approaches that allow
unbiased description and dissection of multi-state RNAs as well as RNA-specific cryo-electron microscopy and
computational approaches for 3D modeling that we believe can unambiguously resolve these structural
controversies. Here, we propose two aims to dissect the complex structural ensemble of the HIV 5′ leader RNA.
First, we will bring to bear a recent four-dimensional chemical mapping method called lock-mutate-map-rescue,
which employs layers of compensatory mutagenesis and helix-locking mutations to detect, stringently test, and
quantitatively establish correlations and anti-correlations between the RNA’s helices and functional elements –
information critical to understanding its multi-state structural ensemble. Second, we will resolve long-range
tertiary contacts and any stereotyped global 3D conformations in the HIV 5’ leader’s structural ensemble though
mutational stabilization, computational modeling, cryo-electron microscopy, and the Multiplexed ·OH Cleavage
Analysis tertiary contact mapping method. We will evaluate the success of this multi-state structural description
through independent tests involving mutations and protein binding events quantitatively evaluated in vitro; and,
later stages, through structure/function experiments, that will test if the HIV 5′ leader transits through the
proposed states in cellulo and in virio.

## Key facts

- **NIH application ID:** 9843110
- **Project number:** 5R21AI145647-02
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Wah Chiu
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $195,625
- **Award type:** 5
- **Project period:** 2019-01-01 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9843110

## Citation

> US National Institutes of Health, RePORTER application 9843110, Structural interrogation of the HIV-1 5′ leader RNA by multidimensional chemical mapping and cryoelectron microscopy (5R21AI145647-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9843110. Licensed CC0.

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