Intrinsic conformational dynamics of the HIV-1 genomic RNA 5′UTR

Abstract

The highly conserved 5′ untranslated region (5′UTR) of the HIV-1 RNA genome is central to the regulation of virus replication. NMR and biochemical experiments support a model in which the 5′UTR can transition between at least two conformational states. In one state the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed, and the genome is competent for kissing loop dimerization and packaging into assembling virions where an extended dimer is formed. According to this model the conformation of the 5′UTR determines the fate of the genome. In this work, the dynamics of this proposed conformational switch and the factors that regulate it were probed using multiple single-molecule and in-gel ensemble FRET assays. Our results show that the HIV-1 5′UTR intrinsically samples conformations that are stabilized by both viral and host factor binding. Annealing of tRNALys3, the primer for initiation of reverse transcription, can promote the kissing dimer but not the extended dimer. In contrast, HIV-1 nucleocapsid (NC) promotes formation of the extended dimer in both the absence and presence of tRNALys3. Our data are consistent with an ordered series of events that involves primer annealing, genome dimerization, and virion assembly.

A New Approach to 3D Modeling of Inhomogeneous Populations of Viral Regulatory RNA

“…The Dimer 3D tertiary prediction placed TAR (light blue) and PolyA (navy) helices stacking on one another ( Figure 2 A; Movie 1). These results agree with published results of chemical mapping [ 8 , 10 , 39 , 40 ] and SAXS [ 34 , 35 ]. The 5′-Cap (magenta space-filling atom) lay at the junction of TAR and PolyA helices and the base of the U5 stem near G104-U105.…”

NMR Studies of Retroviral Genome Packaging

“…1D NMR experiments revealed that the extended interface forms rapidly (within ~10 min) and does not require the presence of NC or other RNA chaperones. More recent single molecule FRET experiments with a 5′-leader fragment containing native U5 and AUG sequences are consistent with the U5: AUG base paired structure observed by NMR, and indicate that the RNA exists as an equilibrium mixture of structures [ 292 ].…”

Illuminating the virus life cycle with single-molecule FRET imaging

“…smFRET has previously provided critical insights into the dynamics of viral proteins throughout their life cycle (Abbondanzieri et al, 2008;Coey et al, 2016;Liu et al, 2008bLiu et al, , 2010. Here we focused on recent applications particularly in the Mothes as well as the Munro laboratories that have extended these applications to physiologically relevant conditions such as complete HIV virions (Brigham et al, 2019;Das et al, 2018;Lu et al, 2019;Ma et al, 2018;Munro et al, 2014a,b). smFRET identified a new conformational Env state on the surface of HIV-1 (Lu et al, 2019), detected and characterized novel structural intermediates in the activation of both HIV-1 and influenza A viral glycoproteins (Das et al, 2018;Ma et al, 2018), and identified a new conformational state of Env that is highly vulnerable to ADCC (Alsahafi et al, 2019).…”

“…Live cell imaging has revealed that assembly and genome packaging are coordinated whereby the genome stimulates the nucleation of capsid assembly (Jouvenet et al, 2009). Both Gag and RNA are conformationally dynamic and smFRET studies have provided insights into both processes (Beerens et al, 2013;Brigham et al, 2019;Coey et al, 2018;Jouvenet et al, 2009;Munro et al, 2014b).…”

“…In the case of the murine leukemia virus, NC has a higher affinity for the dimerized genome (Miyazaki et al, 2010). In the case of HIV, smFRET and bulk in-gel FRET experiments have recently helped to elucidate how tRNA and NC promote transition to an extended dimer (Brigham et al, 2019). Intra-molecular conformational transitions within a single monomer were measuredwith fluorophores positioned at the termini of a 238-nucleotide RNA segment, which contains the Psi packaging signal as well as the primer binding site for the tRNA.…”