Research

14 pages, 1851 KiB

Open AccessArticle

Multifaceted Aspects of HIV-1 Nucleocapsid Inhibition by TAR-Targeting Peptidyl-Anthraquinones Bearing Terminal Aromatic Moieties

by Alice Sosic, Francesco Frecentese, Giulia Olivato, Daniele Rollo, Caterina Carraro, Elia Gamba, Vincenzo Santagada and Barbara Gatto

Viruses 2022, 14(10), 2133; https://0-doi-org.brum.beds.ac.uk/10.3390/v14102133 - 27 Sep 2022

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Abstract

2,6-dipeptidyl-anthraquinones are polycyclic planar systems substituted at opposite ring positions by short aminoacyl side chains. Derivatives with positively charged terminal amino acids showed in vitro inhibition of HIV-1 nucleocapsid (NC) protein correlating with threading intercalation through nucleic acid substrates. We found that the [...] Read more.

2,6-dipeptidyl-anthraquinones are polycyclic planar systems substituted at opposite ring positions by short aminoacyl side chains. Derivatives with positively charged terminal amino acids showed in vitro inhibition of HIV-1 nucleocapsid (NC) protein correlating with threading intercalation through nucleic acid substrates. We found that the variation of the terminal amino acid into an aromatic moiety has profound effects on the NC inhibition of TAR–RNA melting, granting enhanced interaction with the protein. While all compounds showed appreciable NC and TAR binding, they exhibited different strengths driven by the length of the peptidyl side chains and by the stereochemistry of the terminal tyrosine. Unexpectedly, the best inhibitors of NC-induced TAR melting, characterized by the D- configuration of tyrosine, were able to form ternary complexes without competing with TAR–NC recognition sites, as shown by native mass spectrometry experiments. Furthermore, the hydrophobicity of the terminal residue enhances membrane permeation, with positive implications for further studies on these NC–TAR-targeted compounds. Full article

(This article belongs to the Special Issue Retroviral Nucleocapsid Proteins)

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21 pages, 3172 KiB

Open AccessArticle

Upstream of N-Ras (Unr/CSDE1) Interacts with NCp7 and Gag, Modulating HIV-1 IRES-Mediated Translation Initiation

by Nedal Taha, Sarwat Zgheib, Kamal Kant Sharma, Nicolas Humbert, Emmanuel Boutant, Pascal Didier, Yves Mély and Eleonore Real

Viruses 2022, 14(8), 1798; https://0-doi-org.brum.beds.ac.uk/10.3390/v14081798 - 17 Aug 2022

Cited by 1 | Viewed by 1641

Abstract

The Human Immunodeficiency Virus-1 (HIV-1) nucleocapsid protein (NC) as a mature protein or as a domain of the Gag precursor plays important roles in the early and late phases of the infection. To better understand its roles, we searched for new cellular partners [...] Read more.

The Human Immunodeficiency Virus-1 (HIV-1) nucleocapsid protein (NC) as a mature protein or as a domain of the Gag precursor plays important roles in the early and late phases of the infection. To better understand its roles, we searched for new cellular partners and identified the RNA-binding protein Unr/CSDE1, Upstream of N-ras, whose interaction with Gag and NCp7 was confirmed by co-immunoprecipitation and FRET-FLIM. Unr interaction with Gag was found to be RNA-dependent and mediated by its NC domain. Using a dual luciferase assay, Unr was shown to act as an ITAF (IRES trans-acting factor), increasing the HIV-1 IRES-dependent translation. Point mutations of the HIV-1 IRES in a consensus Unr binding motif were found to alter both the IRES activity and its activation by Unr, suggesting a strong dependence of the IRES on Unr. Interestingly, Unr stimulatory effect is counteracted by NCp7, while Gag increases the Unr-promoted IRES activity, suggesting a differential Unr effect on the early and late phases of viral infection. Finally, knockdown of Unr in HeLa cells leads to a decrease in infection by a non-replicative lentivector, proving its functional implication in the early phase of viral infection. Full article

(This article belongs to the Special Issue Retroviral Nucleocapsid Proteins)

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13 pages, 1786 KiB

Open AccessArticle

Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain

by Assia Mouhand, Loussiné Zargarian, Anissa Belfetmi, Marjorie Catala, Marco Pasi, Ewen Lescop, Carine Tisné and Olivier Mauffret

Viruses 2022, 14(3), 632; https://0-doi-org.brum.beds.ac.uk/10.3390/v14030632 - 18 Mar 2022

Cited by 1 | Viewed by 1565

Abstract

The nucleocapsid domain (NCd), located at the C-terminus of the HIV-1 Gag protein, is involved in numerous stages of the replication cycle, such as the packaging of the viral genome and reverse transcription. It exists under different forms through the viral life cycle, [...] Read more.

The nucleocapsid domain (NCd), located at the C-terminus of the HIV-1 Gag protein, is involved in numerous stages of the replication cycle, such as the packaging of the viral genome and reverse transcription. It exists under different forms through the viral life cycle, depending on the processing of Gag by the HIV-1 protease. NCd is constituted of two adjacent zinc knuckles (ZK1 and ZK2), separated by a flexible linker and flanked by disordered regions. Here, conformational equilibria between a major and two minor states were highlighted exclusively in ZK2, by using CPMG and CEST NMR experiments. These minor states appear to be temperature dependent, and their populations are highest at physiological temperature. These minor states are present both in NCp7, the mature form of NCd, and in NCp9 and NCp15, the precursor forms of NCd, with increased populations. The role of these minor states in the targeting of NCd by drugs and its binding properties is discussed. Full article

(This article belongs to the Special Issue Retroviral Nucleocapsid Proteins)

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14 pages, 3195 KiB

Open AccessArticle

RNA Structural Requirements for Nucleocapsid Protein-Mediated Extended Dimer Formation

by Françoise Chaminade, Jean-Luc Darlix and Philippe Fossé

Viruses 2022, 14(3), 606; https://0-doi-org.brum.beds.ac.uk/10.3390/v14030606 - 15 Mar 2022

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Abstract

Retroviruses package two copies of their genomic RNA (gRNA) as non-covalently linked dimers. Many studies suggest that the retroviral nucleocapsid protein (NC) plays an important role in gRNA dimerization. The upper part of the L3 RNA stem-loop in the 5′ leader of the [...] Read more.

Retroviruses package two copies of their genomic RNA (gRNA) as non-covalently linked dimers. Many studies suggest that the retroviral nucleocapsid protein (NC) plays an important role in gRNA dimerization. The upper part of the L3 RNA stem-loop in the 5′ leader of the avian leukosis virus (ALV) is converted to the extended dimer by ALV NC. The L3 hairpin contains three stems and two internal loops. To investigate the roles of internal loops and stems in the NC-mediated extended dimer formation, we performed site-directed mutagenesis, gel electrophoresis, and analysis of thermostability of dimeric RNAs. We showed that the internal loops are necessary for efficient extended dimer formation. Destabilization of the lower stem of L3 is necessary for RNA dimerization, although it is not involved in the linkage structure of the extended dimer. We found that NCs from ALV, human immunodeficiency virus type 1 (HIV-1), and Moloney murine leukemia virus (M-MuLV) cannot promote the formation of the extended dimer when the apical stem contains ten consecutive base pairs. Five base pairs correspond to the maximum length for efficient L3 dimerization induced by the three NCs. L3 dimerization was less efficient with M-MuLV NC than with ALV NC and HIV-1 NC. Full article

(This article belongs to the Special Issue Retroviral Nucleocapsid Proteins)

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21 pages, 4207 KiB

Open AccessArticle

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

by Helena Gien, Michael Morse, Micah J. McCauley, Jonathan P. Kitzrow, Karin Musier-Forsyth, Robert J. Gorelick, Ioulia Rouzina and Mark C. Williams

Viruses 2022, 14(2), 235; https://0-doi-org.brum.beds.ac.uk/10.3390/v14020235 - 25 Jan 2022

Cited by 6 | Viewed by 4058

Abstract

The HIV-1 nucleocapsid protein (NC) is a multi-functional protein necessary for viral replication. Recent studies have demonstrated reverse transcription occurs inside the fully intact viral capsid and that the timing of reverse transcription and uncoating are correlated. How a nearly 10 kbp viral [...] Read more.

The HIV-1 nucleocapsid protein (NC) is a multi-functional protein necessary for viral replication. Recent studies have demonstrated reverse transcription occurs inside the fully intact viral capsid and that the timing of reverse transcription and uncoating are correlated. How a nearly 10 kbp viral DNA genome is stably contained within a narrow capsid with diameter similar to the persistence length of double-stranded (ds) DNA, and the role of NC in this process, are not well understood. In this study, we use optical tweezers, fluorescence imaging, and atomic force microscopy to observe NC binding a single long DNA substrate in multiple modes. We find that NC binds and saturates the DNA substrate in a non-specific binding mode that triggers uniform DNA self-attraction, condensing the DNA into a tight globule at a constant force up to 10 pN. When NC is removed from solution, the globule dissipates over time, but specifically-bound NC maintains long-range DNA looping that is less compact but highly stable. Both binding modes are additionally observed using AFM imaging. These results suggest multiple binding modes of NC compact DNA into a conformation compatible with reverse transcription, regulating the genomic pressure on the capsid and preventing premature uncoating. Full article

(This article belongs to the Special Issue Retroviral Nucleocapsid Proteins)

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24 pages, 5964 KiB

Open AccessArticle

HIV-1 Packaging Visualised by In-Gel SHAPE

by Aaron R. D’Souza, Dhivya Jayaraman, Ziqi Long, Jingwei Zeng, Liam J. Prestwood, Charlene Chan, Dennis Kappei, Andrew M. L. Lever and Julia C. Kenyon

Viruses 2021, 13(12), 2389; https://0-doi-org.brum.beds.ac.uk/10.3390/v13122389 - 29 Nov 2021

Cited by 3 | Viewed by 1944

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Retroviral Nucleocapsid Proteins)

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26 pages, 9096 KiB

Open AccessArticle

The HIV-1 Nucleocapsid Regulates Its Own Condensation by Phase-Separated Activity-Enhancing Sequestration of the Viral Protease during Maturation

by Sébastien Lyonnais, S. Kashif Sadiq, Cristina Lorca-Oró, Laure Dufau, Sara Nieto-Marquez, Tuixent Escribà, Natalia Gabrielli, Xiao Tan, Mohamed Ouizougun-Oubari, Josephine Okoronkwo, Michèle Reboud-Ravaux, José Maria Gatell, Roland Marquet, Jean-Christophe Paillart, Andreas Meyerhans, Carine Tisné, Robert J. Gorelick and Gilles Mirambeau

Viruses 2021, 13(11), 2312; https://0-doi-org.brum.beds.ac.uk/10.3390/v13112312 - 19 Nov 2021

Cited by 7 | Viewed by 3867

Abstract

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular ‘sponges’, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 [...] Read more.

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular ‘sponges’, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak–strong–moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules. Full article

(This article belongs to the Special Issue Retroviral Nucleocapsid Proteins)

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11 pages, 1105 KiB

Open AccessCommunication

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

by Ying Wang, Chao Guo, Xing Wang, Lianmei Xu, Rui Li and Jinzhong Wang

Viruses 2021, 13(10), 1922; https://0-doi-org.brum.beds.ac.uk/10.3390/v13101922 - 24 Sep 2021

Cited by 3 | Viewed by 1581

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Retroviral Nucleocapsid Proteins)

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