# Mechanism of selective packaging of primer tRNALys3 by HIV-1

> **NIH NIH R21** · YALE UNIVERSITY · 2021 · $217,898

## Abstract

Project Summary:
 HIV-1 and the AIDS pandemic remains a significant threat to public health. The lack of a vaccine against
HIV-1 and the looming specter of drug-resistant mutations urge the search for novel drug targets. A critical step
of the HIV-1 life cycle is the selective packaging of host tRNALys1,2,3 during virion assembly, wherein the tRNALys3
isoacceptor is later used as a primer for initiating reverse transcription. This phenomenon of selective tRNALys
packaging by HIV-1 was discovered nearly three decades ago, but the structural and molecular details of this
event remain elusive. LysRS (lysyl-tRNA synthetase), the cellular enzyme for aminoacylation of tRNALys3, has
been shown to play a crucial role in tRNALys packaging by HIV-1. LysRS is predominantly sequestered as part
of the multi-tRNA synthetase complex (MSC) but a catalytically inactive S207 phosphorylated (pS207) pool of
cellular LysRS is released from the MSC upon HIV-1 infection. HIV-1 Gag plays the primary role in LysRS and
tRNALys3 packaging. The current model postulates that tRNALys3 is indirectly packaged by HIV-1 Gag via an
interaction between its capsid protein (CA) and the catalytic domain of LysRS. However, this model is based
primarily on studies carried out with unmodified LysRS in the absence of tRNALys3 and our preliminary data
contradicts this model. HIV-1 has been shown to selectively package uncharged tRNALys3 over charged tRNALys3
and pS207-LysRS has been proposed to play a role in this packaging bias. There is no high-resolution structural
information available on pS207-LysRS to provide a mechanistic basis for its lack of aminoacylation activity, its
interactions with HIV-1 Gag, or the non-canonical functions it performs. The lack of any structural information on
how HIV-1 Gag recruits LysRS and tRNALys3 has precluded targeting this step for drug development and the
proposed work will address this knowledge gap. The overall goal of this research is to establish a mechanistic
and structural basis for selective packaging of uncharged tRNALys3 by HIV-1. In Aim 1, we will carry out a
comprehensive comparative characterization of the HIV-1 factors reported to bind LysRS and tRNALys3 using
qualitative and quantitative biochemical and biophysical techniques. The comparative molecular dissection will
allow us to establish the minimal core components of the tRNALys3 packaging complex. We will use a
phosphomimetic mutant of LysRS (LysRSS207D) to study the role of pS207-LysRS in selective packaging of
uncharged tRNALys3 and study the kinetics of the ternary tRNALys3 packaging complex to probe any cooperativity
in complex formation. The thorough biochemical characterization will help establish a stable ternary tRNALys3
packaging complex for structural studies. In Aim 2, we will use a parallel two-pronged approach of single particle
cryo-EM and X-ray crystallography to determine a high-resolution structure of the ternary tRNALys3 packaging
complex and validate our structura...

## Key facts

- **NIH application ID:** 10258167
- **Project number:** 1R21AI157890-01A1
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Karin M Musier-Forsyth
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $217,898
- **Award type:** 1
- **Project period:** 2021-03-22 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10258167

## Citation

> US National Institutes of Health, RePORTER application 10258167, Mechanism of selective packaging of primer tRNALys3 by HIV-1 (1R21AI157890-01A1). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10258167. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*