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...