scholarly journals HIV-1 Packaging Visualised by In-Gel SHAPE

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2389
Author(s):  
Aaron R. D’Souza ◽  
Dhivya Jayaraman ◽  
Ziqi Long ◽  
Jingwei Zeng ◽  
Liam J. Prestwood ◽  
...  
Keyword(s):  
Rna Structure ◽  
Structural Changes ◽  
Structural Protein ◽  
Packaging Signal ◽  
Packaging Process ◽  
Gag Polyprotein ◽  
Rna Conformation ◽  
Shape Selective ◽  
Insight Into ◽  
Hiv 1

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.

scholarly journals Potential Achilles heels of SARS-CoV-2 displayed by the base order-dependent component of RNA folding energy

2020 ◽  
Author(s):  
Chiyu Zhang ◽  
Donald R. Forsdyke
Keyword(s):  
Rna Structure ◽  
Folding Energy ◽  
Packaging Signal ◽  
Antiviral Compound ◽  
N Protein ◽  
Gag Polyprotein ◽  
Protein Encoding ◽  
Dependent Component ◽  
High Base ◽  
Hiv 1

ABSTRACTBase order, not composition, best reflects local evolutionary pressure for folding of single-stranded nucleic acids. The base order-dependent component of folding energy has revealed a highly conserved region in HIV-1 genomes that associates with RNA structure. This corresponds to a packaging signal that is recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 “Achilles heel,” the signal can be targeted by a recently described antiviral compound (NSC 260594) or by synthetic oligonucleotides. Thus, a conserved base-order-rich region of HIV-1 may facilitate therapeutic attack. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e.g. NSP3, ORF3a), we suggest that their nucleic acid level functions – such as the ribosomal frameshifting element (FSE) that facilitates differential expression of 1a and 1ab polyproteins – can be considered potential “Achilles heels” for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. The region of the FSE scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.

The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the gag polyprotein

2000 ◽  
Vol 301 (5) ◽  
pp. 1315
Author(s):  
Amanda Zeffman ◽  
Stuart Hassard ◽  
Gabriele Varani ◽  
Andrew Lever
Keyword(s):  
Packaging Signal ◽  
Stem Loop ◽  
Gag Polyprotein ◽  
Hiv 1

scholarly journals MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat

2018 ◽  
Vol 115 (52) ◽  
pp. 13258-13263 ◽  
Author(s):  
Michael D. Purdy ◽  
Dan Shi ◽  
Jakub Chrustowicz ◽  
Johan Hattne ◽  
Tamir Gonen ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Electrostatic Interactions ◽  
Hydrophobic Interactions ◽  
Binding Interactions ◽  
Structure Based Drug Design ◽  
Gag Polyprotein ◽  
Pentacyclic Triterpenoid ◽  
Maturation Inhibitor ◽  
Insight Into ◽  
Hiv 1

HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.

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

Retrovirology ◽  
2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Shuohui Liu ◽  
Pratibha C. Koneru ◽  
Wen Li ◽  
Chathuri Pathirage ◽  
Alan N. Engelman ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Structural Changes ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Virus Particles ◽  
Structure Changes ◽  
Apical Loop ◽  
Viral Genomic Rna ◽  
Hiv 1

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

scholarly journals Three-Dimensional RNA Structure of the Major HIV-1 Packaging Signal Region

Structure ◽  
2013 ◽  
Vol 21 (6) ◽  
pp. 951-962 ◽  
Author(s):  
James D. Stephenson ◽  
Haitao Li ◽  
Julia C. Kenyon ◽  
Martyn Symmons ◽  
Dave Klenerman ◽  
...  
Keyword(s):  
Rna Structure ◽  
Three Dimensional ◽  
Packaging Signal ◽  
Signal Region ◽  
Hiv 1

scholarly journals 5′-Cap sequestration is an essential determinant of HIV-1 genome packaging

2021 ◽  
Vol 118 (37) ◽  
pp. e2112475118
Author(s):  
Pengfei Ding ◽  
Siarhei Kharytonchyk ◽  
Nansen Kuo ◽  
Emily Cannistraci ◽  
Hana Flores ◽  
...  
Keyword(s):  
Rna Processing ◽  
Binding Sites ◽  
Rna Structure ◽  
Virus Assembly ◽  
Wild Type ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Rna Dimerization ◽  
Gag Polyprotein ◽  
Hiv 1

HIV-1 selectively packages two copies of its 5′-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5′ leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5′ leader. ΨCES lacks a 5′-tandem hairpin element that sequesters the 5′ cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5′ ribozyme to ΨCES to enable cotranscriptional shedding of the 5′ cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5′ cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5′-capped RNA genomes.

scholarly journals The Zinc Content of HIV-1 NCp7 Affects Its Selectivity for Packaging Signal and Affinity for Stem-Loop 3

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1922
Author(s):  
Ying Wang ◽  
Chao Guo ◽  
Xing Wang ◽  
Lianmei Xu ◽  
Rui Li ◽  
...  
Keyword(s):  
Rna Binding ◽  
Zinc Content ◽  
Binding Property ◽  
Zinc Ions ◽  
Packaging Signal ◽  
Stem Loop ◽  
Virus Maturation ◽  
Gag Polyprotein ◽  
Zinc Finger Motifs ◽  
Hiv 1

The nucleocapsid (NC) protein of human immunodeficiency (HIV) is a small, highly basic protein containing two CCHC zinc-finger motifs, which is cleaved from the NC domain of the Gag polyprotein during virus maturation. We previously reported that recombinant HIV-1 Gag and NCp7 overexpressed in an E. coli host contains two and one zinc ions, respectively, and Gag exhibited much higher selectivity for packaging signal (Psi) and affinity for the stem-loop (SL)-3 of Psi than NCp7. In this study, we prepared NCp7 containing 0 (0NCp7), 1 (NCp7) or 2 (2NCp7) zinc ions, and compared their secondary structure, Psi-selectivity and SL3-affinity. Along with the decrease of the zinc content, less ordered conformations were detected. Compared to NCp7, 2NCp7 exhibited a much higher Psi-selectivity and SL3-affinity, similar to Gag, whereas 0NCp7 exhibited a lower Psi-selectivity and SL3-affinity, similar to the H23&H44K double mutant of NCp7, indicating that the different RNA-binding property of Gag NC domain and the mature NCp7 may be resulted, at least partially, from their different zinc content. This study will be helpful to elucidate the critical roles that zinc played in the viral life cycle, and benefit further investigations of the functional switch from the NC domain of Gag to the mature NCp7.

scholarly journals Insight into the HIV-1 Vif SOCS-box–ElonginBC interaction

Open Biology ◽  
2013 ◽  
Vol 3 (11) ◽  
pp. 130100 ◽  
Author(s):  
Zhisheng Lu ◽  
Julien R. C. Bergeron ◽  
R. Andrew Atkinson ◽  
Torsten Schaller ◽  
Dennis A. Veselkov ◽  
...  
Keyword(s):  
Solution Structure ◽  
Hydrophobic Interactions ◽  
Dynamic Information ◽  
Ubiquitin Ligase Complex ◽  
Biophysical Studies ◽  
Viral Infectivity Factor ◽  
Β Sheet ◽  
Socs Box ◽  
Insight Into ◽  
Hiv 1

The HIV-1 viral infectivity factor (Vif) neutralizes cell-encoded antiviral APOBEC3 proteins by recruiting a cellular ElonginB (EloB)/ElonginC (EloC)/Cullin5-containing ubiquitin ligase complex, resulting in APOBEC3 ubiquitination and proteolysis. The suppressors-of-cytokine-signalling-like domain (SOCS-box) of HIV-1 Vif is essential for E3 ligase engagement, and contains a BC box as well as an unusual proline-rich motif. Here, we report the NMR solution structure of the Vif SOCS–ElonginBC (EloBC) complex. In contrast to SOCS-boxes described in other proteins, the HIV-1 Vif SOCS-box contains only one α-helical domain followed by a β-sheet fold. The SOCS-box of Vif binds primarily to EloC by hydrophobic interactions. The functionally essential proline-rich motif mediates a direct but weak interaction with residues 101–104 of EloB, inducing a conformational change from an unstructured state to a structured state. The structure of the complex and biophysical studies provide detailed insight into the function of Vif's proline-rich motif and reveal novel dynamic information on the Vif–EloBC interaction.

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