HIV-1 Packaging Visualised by In-Gel SHAPE.

Aaron R D’Souza,Dennis Kappei,Andrew M L Lever,Ziqi Long,Jingwei Zeng,Julia C Kenyon,Dhivya Jayaraman,Liam J Prestwood,Charlene Chan

doi:10.3390/v13122389

Abstract

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA–protein complex. Here, we apply the in-gel SHAPE (selective 2’OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag–dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.

Highlights

Introduction published maps and institutional affilThe HIV-1 genome is a single-stranded RNA molecule that encodes the essential structural polyproteins Gag, Gag-Pol and the envelope glycoprotein Env, together with a number of accessory factors that aid viral replication and immune evasion. gRNA serves as a template for the translation of the viral structural proteins found in Gag and the enzymes encoded by Pol, as well as being captured by Gag for packaging into virions
In-Gel SHAPE of an RNA–Protein Complex Accurately Reports upon Its RNA Structure
Firstly, the suitability of in-gel SHAPE to evaluate RNA structural changes caused by protein binding was investigated

Summary

Introduction

Introduction published maps and institutional affilThe HIV-1 genome (gRNA) is a single-stranded RNA molecule that encodes the essential structural polyproteins Gag, Gag-Pol and the envelope glycoprotein Env, together with a number of accessory factors that aid viral replication and immune evasion. gRNA serves as a template for the translation of the viral structural proteins found in Gag and the enzymes encoded by Pol, as well as being captured by Gag for packaging into virions. The HIV-1 genome (gRNA) is a single-stranded RNA molecule that encodes the essential structural polyproteins Gag, Gag-Pol and the envelope glycoprotein Env, together with a number of accessory factors that aid viral replication and immune evasion. GRNA serves as a template for the translation of the viral structural proteins found in Gag and the enzymes encoded by Pol, as well as being captured by Gag for packaging into virions. The gRNA undergoes dimerisation, resulting in two copies of the genome being encapsidated into the budding virions. Gag and Gag-Pol polyproteins are cleaved into their individual components—matrix (MA), capsid (CA) and nucleocapsid (NC)—as well as three smaller peptides [1], and the enzymes reverse transcriptase (RT), integrase (IN) and protease (PR) by PR itself. Upon infection of a new cell, RT initiates the reverse transcription of the ssRNA genome into dsDNA using a cellular tRNALys primer that anneals to the gRNA at some stage during viral assembly iations.

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Conclusion