Abstract
BackgroundDuring 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.ResultsUsing 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.ConclusionsOverall, 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
Highlights
During HIV-1 maturation, Gag and Gag-Pol polyproteins are proteolytically cleaved and the capsid protein polymerizes to form the honeycomb capsid lattice
Many class II IN mutations lead to aberrant virion morphogenesis and mis-localization of the viral ribonucleoprotein complex (vRNP) outside the capsid core, resulting in non-infectious virions [5, 7,8,9,10,11,12,13]
We applied crosslinking-coupled selective 2′-hydroxyl acylation analyzed by primer extension (XL-SHAPE), a single-nt resolution technique that allows the identification of both direct protein interaction sites and protein binding-induced RNA conformational changes [40]
Summary
During HIV-1 maturation, Gag and Gag-Pol polyproteins are proteolytically cleaved and the capsid protein polymerizes to form the honeycomb capsid lattice. IN and NC were previously demonstrated to bind to the gRNA in an orthogonal manner in virio; the effect of IN binding alone or simultaneous binding of both proteins on gRNA structure is not yet well understood Retroviruses such as HIV-1 undergo dramatic morphological changes during viral assembly and maturation that are essential to form mature infectious virions [1, 2]. Immature HIV-1 particles undergo maturation wherein the polyproteins are cleaved by the viral protease and rearrange to form the infectious viral particle [3] During this step, liberated capsid (CA) proteins assemble to form the mature capsid core, enclosing a viral ribonucleoprotein complex (vRNP) containing two copies of genomic RNA (gRNA) that are coated and condensed by nucleocapsid (NC) proteins; the vRNP contains reverse transcriptase (RT) and integrase (IN) [1, 3]. Supplying IN in trans to class II mutant virions partially restored vRNP encapsidation and HIV-1 infectivity, suggesting an active role for IN in HIV-1 particle morphogenesis [11]
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