Significant Differences in RNA Structure Destabilization by HIV-1 GagDp6 and NCp7 Proteins.

Micah J Mccauley,Mark C Williams,Ioulia Rouzina,Jasmine Li,Megan E Núñez

doi:10.3390/v12050484

Micah J Mccauley, Mark C Williams + Show 3 more

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https://doi.org/10.3390/v12050484

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Abstract

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play distinct roles in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structures, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a complementary RNA hairpin. In contrast, during viral assembly, NC, as a domain of the group-specific antigen (Gag) polyprotein, binds the genomic RNA and facilitates packaging into new virions. It is not clear how the same protein, alone or as part of Gag, performs such different RNA binding functions in the viral life cycle. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium stability and unfolding barrier for TAR RNA. Comparing measured results with a model of discrete protein binding allows us to localize affected binding sites, in addition to quantifying hairpin stability. We find that, while both NCp7 and Gag∆p6 destabilize the TAR hairpin, Gag∆p6 binding is localized to two sites in the stem, while NCp7 targets sites near the top loop. Unlike Gag∆p6, NCp7 destabilizes this loop, shifting the location of the reaction barrier toward the folded state and increasing the natural rate of hairpin opening by ~104. Thus, our results explain why Gag cleavage and NC release is an essential prerequisite for reverse transcription within the virion.

Highlights

DNA and RNA hairpins participate in many critical rate-limiting biochemical processes [1,2,3,4,5].These sequences are often chaperoned by ligands that facilitate the formation of a diverse set of secondary and tertiary structures to suit their roles [2,3,4,5,6,7,8]
Parameters extracted from optical tweezer experiments are analyzed to create a landscape of transactivation response (TAR)
Gag∆p6 binding is directly compared to the ability of NCp7 to dramatically enhance the opening rate of the TAR hairpin, which was shown in our previous work [23]

Summary

Introduction

DNA and RNA hairpins participate in many critical rate-limiting biochemical processes [1,2,3,4,5]. These sequences are often chaperoned by ligands that facilitate the formation of a diverse set of secondary and tertiary structures to suit their roles [2,3,4,5,6,7,8]. The transactivation response (TAR) sequence in the HIV-1 virion forms a hairpin, which is involved in several key steps in the virion life cycle (Figure 1A), including transcription and reverse transcription [9,10]. Once the virion infects a new host, the hairpin must open to facilitate the key step of minus strand transfer (Figure 1C) [11,12,13,14,15,16]

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