HIV-1 integrase binding to genomic RNA 5′-UTR induces local structural changes in vitro and in virio

Abstract

Background During HIV-1 maturation, Gag and Gag-Pol polyproteins are proteolytically cleaved and the capsid protein polymerizes to form the honeycomb capsid lattice. HIV-1 integrase (IN) binds the viral genomic RNA (gRNA) and impairment of IN-gRNA binding leads to mis-localization of the nucleocapsid protein (NC)-condensed viral ribonucleoprotein complex outside the capsid core. IN and NC were previously demonstrated to bind to the gRNA in an orthogonal manner in virio; however, the effect of IN binding alone or simultaneous binding of both proteins on gRNA structure is not yet well understood. Results Using crosslinking-coupled selective 2′-hydroxyl acylation analyzed by primer extension (XL-SHAPE), we characterized the interaction of IN and NC with the HIV-1 gRNA 5′-untranslated region (5′-UTR). NC preferentially bound to the packaging signal (Psi) and a UG-rich region in U5, irrespective of the presence of IN. IN alone also bound to Psi but pre-incubation with NC largely abolished this interaction. In contrast, IN specifically bound to and affected the nucleotide (nt) dynamics of the apical loop of the transactivation response element (TAR) and the polyA hairpin even in the presence of NC. SHAPE probing of the 5′-UTR RNA in virions produced from allosteric IN inhibitor (ALLINI)-treated cells revealed that while the global secondary structure of the 5′-UTR remained unaltered, the inhibitor treatment induced local reactivity differences, including changes in the apical loop of TAR that are consistent with the in vitro results. Conclusions Overall, the binding interactions of NC and IN with the 5′-UTR are largely orthogonal in vitro. This study, together with previous probing experiments, suggests that IN and NC binding in vitro and in virio lead to only local structural changes in the regions of the 5′-UTR probed here. Accordingly, disruption of IN-gRNA binding by ALLINI treatment results in local rather than global secondary structure changes of the 5′-UTR in eccentric virus particles. Graphical Abstract

HIV-1 Nucleocapsid Protein Binds Double-Stranded DNA in Multiple Modes to Regulate Compaction and Capsid Uncoating

“…further experiments both in vitro and in infected cells. The role of other DNA binding proteins, such as IN, which may affect NC condensation, should also be investigated [70,71].…”

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

“…However, early expression of Gag/Pol could be important for the replication cycle in addition to their canonical roles at late replication steps. For instance, Integrase is known to interact with TAR RNA and Tat, and has been recently shown to regulate proviral DNA transcription at early time points of infection (Elliott et al, 2020; Liu et al, 2021; Rocchi et al, 2021; Winans and Goff, 2020).…”

The C-Terminal Domain of HIV-1 Integrase: A Swiss Army Knife for the Virus?

“…This interaction allows the tail region to act as a sensor of the proper TAR structure (https: //doi.org/10.1101/2021.10.21.465253, accessed on 31 May 2022). Intriguingly, the binding of IN induces conformational changes of a TAR apical hexaloop overexposing a critical nucleotide (https://doi.org/10.1101/2021.10.21.465253, accessed on 31 May 2022; [123]). A similar conformation of TAR has been observed in the structure of the TAR:Tat:SEC -core complex, allowing the binding of Cyclin T1 to TAR for a proviral transcription boost [120].…”

“…The presence of the bulge and the loop in TAR RNA, rather than its sequence, is critical for IN binding, as the deletion of one or both markedly decreases the binding affinity of full-length protein [ 7 ]. A crosslinking-coupled SHAPE assay using the IN:LEDGF/p75 IBD complex and the whole HIV-1 5′UTR revealed a crosslinking site located on C39, near the bulge region of TAR [ 123 ] ( Figure 7 A).…”