Structural maturation of the HIV-1 RNA 5’ untranslated region by Pr55 ^Gag and its maturation products

Abstract

Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55 Gag precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55 Gag or its maturation products (NCp15, NCp9 and NCp7) and the 5ʹ gRNA region and their structural consequences, in vitro . We show that Pr55 Gag and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55 Gag maturation products on the gRNA structural maturation.

Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments

“…However, any protein that shows activity in any of the assays designed to test RNA chaperone activity [ 41 ] is usually considered an RNA chaperone even though its binding to RNA is not transient. Over time, numerous structural [ 31 , 33 , 42 , 43 ] and non-structural [ 32 , 44 ] viral proteins have been considered RNA chaperones, even though most of them do not spontaneously dissociate from RNA. The effect of such RNA chaperones can be assessed by comparing the RNA structure before the addition of the protein and after the removal of the protein by SDS or/and proteinase K treatment [ 27 , 32 , 42 ].…”

“…Initially, RNA chaperones were defined as proteins that transiently bind to RNA and remodel its structure [ 41 , 54 ]. However, many viral proteins that remain associated with the RNA to which they bind were found to affect the RNA structure when they were removed by protease degradation and/or phenol/chloroform extraction, and by extension, are also considered as RNA chaperones [ 31 , 32 , 33 , 42 ]. During IAV replication, the nascent vRNA and complementary RNA associate with NP as soon as they are synthesized, and NP is transiently displaced from the vRNA template during replication and transcription [ 2 , 55 , 56 ].…”

Extensive uORF translation from HIV-1 transcripts conditions DDX3 dependency for expression of main ORFs and elicits specific T cell immune responses in infected individuals

“…As a final confirmation of these results, we created reporter constructs consisting of the 5’UTR of the HIV-1 genomic RNA, a mutant version where most non-AUG initiation codons were replaced by UAG stop codons (Figure 6B, see No-uORFs 5’UTR) and a second mutant where 4 of the putative non-AUG initiation codons were replaced by AUG codons (Figure 6B, see AUG-uORFs 5’UTR), followed by the renilla luciferase coding sequence. Compensatory mutations were introduced in mutant 5’UTRs in order to maintain their overall secondary structure, which was probed by SHAPE (Gilmer et al, 2022; Merino et al, 2005; Wilkinson et al, 2006) (Supplementary Figure 5A and B). These reporter plasmids were in vitro transcribed to generate capped and polyadenylated RNAs that were transfected into HEK293T cells.…”

Structural and computational studies of HIV-1 RNA

“…It was further confirmed that the internal loop of SL1 was the primary recognition site for Gag among various spliced and unspliced RNAs [ 227 ]. Furthermore, Gag polyprotein and its NC-containing maturation products were shown to bind at different gRNA sites with distinct contributions to the gRNA structural maturation [ 228 ]. Isothermal titration calorimetry (ITC) was used to show that major interactions between HIV-1 gRNA and Gag formed during virion assembly were energetically favourable [ 229 ].…”