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

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.

Modulation of the HIV nucleocapsid dynamics finely tunes its RNA-binding properties during virion genesis.

During HIV-1 assembly and budding, Gag protein, in particular the C-terminal domain containing the nucleocapsid domain (NCd), p1 and p6, is the site of numerous interactions with viral and cellular factors. Most in vitro studies of Gag have used constructs lacking p1 and p6. Here, using NMR spectroscopy, we show that the p1-p6 region of Gag (NCp15) is largely disordered, but interacts transiently with the NCd. These interactions modify the dynamic properties of the NCd. Indeed, using isothermal titration calorimetry (ITC), we have measured a higher entropic penalty to RNA-binding for the NCd precursor, NCp15, than for the mature form, NCp7, which lacks p1 and p6. We propose that during assembly and budding of virions, concomitant with Gag oligomerization, transient interactions between NCd and p1-p6 become salient and responsible for (i) a higher level of structuration of p6, which favours recruitment of budding partners; and (ii) a higher entropic penalty to RNA-binding at specific sites that favours non-specific binding of NCd at multiple sites on the genomic RNA (gRNA). The contributions of p6 and p1 are sequentially removed via proteolysis during Gag maturation such that the RNA-binding specificity of the mature protein is governed by the properties of NCd.

Equilibrium Model of Drug-Modulated GagPol-Embedded HIV-1 Reverse Transcriptase Dimerization to Enhance Premature Protease Activation.

Lack of effective strategies for killing cells latently infected with HIV-1 limits the eradication of AIDS. Unfortunately, current antiretroviral inhibitors are designed to target virus production but not latent infection. Interestingly, some non-nucleoside reverse transcriptase inhibitors (NNRTIs) have shown off-design effects, specifically, premature activation of HIV-1 protease (PR) within virus-infected cells that induces apoptosis. Here, we analyze an equilibrium model of HIV-1 reverse transcriptase (RT) binding to NNRTIs to understand the optimal binding characteristics that enhance RT dimerization within embedded GagPol dimers. This would allow NNRTIs to act as PR autoactivation enhancers (PAEs). We compute that ∼700-fold enhancement is theoretically possible by PAEs. Both a strong drug-dimer binding affinity (KD12 < 100 nM) and relatively weaker drug-monomer affinity (KD2/KD12 > 10) are required for significant enhancement (∼50-fold or more) relative to the drug-free dimer concentration within a drug concentration limit of 10 µM. Our approach rationalizes the observed effects of efavirenz on premature activation of PR and may be useful to guide the design of suitable drug candidates and their optimal dosage regimens for this therapy class.

The Myxobacterial Metabolite Soraphen A Inhibits HIV-1 by Reducing Virus Production and Altering Virion Composition.

Soraphen A is a myxobacterial metabolite that blocks the acetyl-coenzyme A carboxylase of the host and was previously identified as a novel HIV inhibitor. Here, we report that soraphen A acts by reducing virus production and altering the gp120 virion content, impacting entry capacity and infectivity. These effects are partially reversed by addition of palmitic acid, suggesting that inhibition of HIV envelope palmitoylation is one of the mechanisms of antiviral action.

Oligonucleotide-Lipid Conjugates Forming G-Quadruplex Structures Are Potent and Pangenotypic Hepatitis C Virus Entry Inhibitors In Vitro and Ex Vivo.

A hepatitis C virus (HCV) epidemic affecting HIV-infected men who have sex with men (MSM) is expanding worldwide. In spite of the improved cure rates obtained with the new direct-acting antiviral drug (DAA) combinations, the high rate of reinfection within this population calls urgently for novel preventive interventions. In this study, we determined in cell culture and ex vivo experiments with human colorectal tissue that lipoquads, G-quadruplex DNA structures fused to cholesterol, are efficient HCV pangenotypic entry and cell-to-cell transmission inhibitors. Thus, lipoquads may be promising candidates for the development of rectally applied gels to prevent HCV transmission.

Nucleocapsid Protein: A Desirable Target for Future Therapies Against HIV-1.

The currently available anti-HIV-1 therapeutics is highly beneficial to infected patients. However, clinical failures occur as a result of the ability of HIV-1 to rapidly mutate. One approach to overcome drug resistance is to target HIV-1 proteins that are highly conserved among phylogenetically distant viral strains and currently not targeted by available therapies. In this respect, the nucleocapsid (NC) protein, a zinc finger protein, is particularly attractive, as it is highly conserved and plays a central role in virus replication, mainly by interacting with nucleic acids. The compelling rationale for considering NC as a viable drug target is illustrated by the fact that point mutants of this protein lead to noninfectious viruses and by the inability to select viruses resistant to a first generation of anti-NC drugs. In our review, we discuss the most relevant properties and functions of NC, as well as recent developments of small molecules targeting NC. Zinc ejectors show strong antiviral activity, but are endowed with a low therapeutic index due to their lack of specificity, which has resulted in toxicity. Currently, they are mainly being investigated for use as topical microbicides. Greater specificity may be achieved by using non-covalent NC inhibitors (NCIs) targeting the hydrophobic platform at the top of the zinc fingers or key nucleic acid partners of NC. Within the last few years, innovative methodologies have been developed to identify NCIs. Though the antiviral activity of the identified NCIs needs still to be improved, these compounds strongly support the druggability of NC and pave the way for future structure-based design and optimization of efficient NCIs.

A protein ballet around the viral genome orchestrated by HIV-1 reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed RNA to Vpr-bridged DNA.

HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing pre-integration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin.

Features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function.

Retroviral nucleocapsid (NC) is central to viral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NC's aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting in the condensation of the nucleocapsid within the capsid shell. Reverse transcription also depends on NC processing, whereas this process provokes NC dissociation from double-stranded DNA, leading to a preintegration complex (PIC), competent for host chromosomal integration. In addition NC interacts with cellular proteins, some of which are involved in viral budding, and also with several viral proteins. All of these properties are reviewed here, focusing on HIV-1 as a paradigmatic reference and highlighting the plasticity of the nucleocapsid architecture.

Rad51 polymerization reveals a new chromatin remodeling mechanism.

Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics.

[How proviral DNA is integrated into the host cell DNA and how this process can be inhibited]. / Cómo se integra el ADN proviral en el ADN de la célula del huésped y cómo se puede inhibir el proceso.

The HIV replication cycle passes through a stage of integrating proviral DNA into the cell's DNA. In this process, the viral enzyme, integrase, catalyses two reactions. The first reaction, which seems to occur in the cytoplasm, involves 3'-end processing, in which two nucleotides are removed from the 3' ends of the viral DNA by integrase. The second reaction, which occurs in the nucleus, involves the strand transfer reaction, catalyzed by integrase, in which the recessed 3' ends of the viral DNA are joined to the protruding 5' ends in the target DNA. Although this activity has not yet been completely defined and the structure of the active form of integrase, probably a tetramer, has not been resolved, drugs of the diketoacid (DKA) family have been found. These drugs are highly potent inhibitors of the second phase, the strand transfer reaction. Through a series of optimizations, a highly effective molecule for clinical use, raltegravir, has been achieved. The present article provides a summary of basic knowledge on integrase, as well as the activity and the modes of inhibition of this enzyme. Also discussed is the reduced, but nevertheless real, development of resistance to raltegravir, requiring second-generation integrase inhibitors to be designed.

HIV-1 protease and reverse transcriptase control the architecture of their nucleocapsid partner.

The HIV-1 nucleocapsid is formed during protease (PR)-directed viral maturation, and is transformed into pre-integration complexes following reverse transcription in the cytoplasm of the infected cell. Here, we report a detailed transmission electron microscopy analysis of the impact of HIV-1 PR and reverse transcriptase (RT) on nucleocapsid plasticity, using in vitro reconstitutions. After binding to nucleic acids, NCp15, a proteolytic intermediate of nucleocapsid protein (NC), was processed at its C-terminus by PR, yielding premature NC (NCp9) followed by mature NC (NCp7), through the consecutive removal of p6 and p1. This allowed NC co-aggregation with its single-stranded nucleic-acid substrate. Examination of these co-aggregates for the ability of RT to catalyse reverse transcription showed an effective synthesis of double-stranded DNA that, remarkably, escaped from the aggregates more efficiently with NCp7 than with NCp9. These data offer a compelling explanation for results from previous virological studies that focused on i) Gag processing leading to nucleocapsid condensation, and ii) the disappearance of NCp7 from the HIV-1 pre-integration complexes. We propose that HIV-1 PR and RT, by controlling the nucleocapsid architecture during the steps of condensation and dismantling, engage in a successive nucleoprotein-remodelling process that spatiotemporally coordinates the pre-integration steps of HIV-1. Finally we suggest that nucleoprotein remodelling mechanisms are common features developed by mobile genetic elements to ensure successful replication.

Molecular modelling studies on the interactions of human DNA topoisomerase IB with pyridoxal-compounds.

Candida guilliermondii and human DNA topoisomerases I are inhibited by PL (pyridoxal), PLP (pyridoxal 5'-phosphate) and PLP-AMP (pyridoxal 5'-diphospho-5'-adenosine) (PL

Transmission electron microscopy reveals an optimal HIV-1 nucleocapsid aggregation with single-stranded nucleic acids and the mature HIV-1 nucleocapsid protein.

HIV-1 nucleocapsid protein (NCp7) condenses the viral RNA within the mature capsid. In a capsid-free system, NCp7 promotes an efficient mechanism of aggregation with both RNA and DNA. Here, we show an analysis of these macromolecular complexes by dark-field imaging using transmission electron microscopy. Thousands of mature NCp7 proteins co-aggregate with hundreds of single-stranded circular DNA molecules (ssDNA) within minutes, as observed with poly(rA). These co-aggregates are highly stable but dynamic structures, as they dissociate under harsh conditions, and after addition of potent ssDNA or NCp7 competitive ligands. The N-terminal domain and zinc fingers of NCp7 are both required for efficient association. Addition of magnesium slightly increases the avidity of NCp7 for ssDNA, while it strongly inhibits co-aggregation with relaxed circular double-stranded DNA (dsDNA). This DNA selectivity is restricted to mature NCp7, compared to its precursors NCp15 and NCp9. Moreover, for NCp15, the linkage of NCp7 with the Gag C-terminal p6-peptide provokes a deficiency in ssDNA aggregation, but results in DNA spreading similar to prototypical SSB proteins. Finally, this co-aggregation is discussed in a dynamic architectural context with regard to the mature HIV-1 nucleocapsid. On the basis of the present data, we propose that condensation of encapsidated RNA requires the C-terminal processing of NCp. Subsequently, disassembly of the nucleocapsid should be favoured once dsDNA is produced by HIV-1 reverse transcriptase.

Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid.

The present results demonstrate that pyridoxal, pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the epsilon-amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (K(i) = 40 microM) that blocks the cleavable complex formation. Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein. The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK*DSIR). Targeted lysine (K*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K532), Candida albicans (K468), Saccharomyces cerevisiae (K458) and Schizosaccharomyces pombe (K505). In the human enzyme, K532, belonging to the active site acts as a general acid catalyst and is therefore essential for activity. The spatial orientation of K532-PLP within the active site was approached by molecular modeling using available crystallographic data. The PLP moiety was found at close proximity of several active residues. PLP could be involved in the cellular control of topoisomerases IB. It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid.

G-quartets direct assembly of HIV-1 nucleocapsid protein along single-stranded DNA.

The d(TTGGGGGGTACAGTGCA) sequence, derived from the human immunodeficiency virus type 1 (HIV-1) central DNA flap, can form in vitro an intermolecular parallel DNA quadruplex. This work demonstrates that the HIV-1 nucleocapsid protein (NCp) exhibits a high affinity (10(8) M(-1)) for this quadruplex. This interaction is predominantly hydrophobic, maintained by a stabilization between G-quartet planes and the C-terminal zinc finger of the protein. It also requires 5 nt long tails flanking the quartets plus both the second zinc-finger and the N-terminal domain of NCp. The initial binding nucleates an ordered arrangement of consecutive NCp along the four single-stranded tails. Such a process requires the N-terminal zinc finger, and was found to occur for DNA site sizes shorter than usual in a sequence-dependent manner. Concurrently, NCp binding is efficient on a G'2 quadruplex also derived from the HIV-1 central DNA flap. Apart from their implication within the DNA flap, these data lead to a model for the nucleic acid architecture within the viral nucleocapsid, where adjacent single-stranded tails and NCp promote a compact assembly of NCp and nucleic acid growing from stably and primary bound NCp.

Contribution of DNA conformation and topology in right-handed DNA wrapping by the Bacillus subtilis LrpC protein.

The Bacillus subtilis LrpC protein belongs to the Lrp/AsnC family of transcriptional regulators. It binds the upstream region of the lrpC gene and autoregulates its expression. In this study, we have dissected the mechanisms that govern the interaction of LrpC with DNA by electrophoretic mobility shift assay, electron microscopy, and atomic force microscopy. LrpC is a structure-specific DNA binding protein that forms stable complexes with curved sequences containing phased A tracts and wraps DNA to form spherical, nucleosome-like structures. Formation of such wraps, initiated by cooperative binding of LrpC to DNA, results from optimal protein/protein interactions specified by the DNA conformation. In addition, we have demonstrated that LrpC constrains positive supercoils by wrapping the DNA in a right-handed superhelix, as visualized by electron microscopy.

G-quartets assembly within a G-rich DNA flap. A possible event at the center of the HIV-1 genome.

Stretches of guanines can associate in vitro through Hoogsteen hydrogen bonding to form four-stranded structures. In the HIV-1 central DNA flap, generated by reverse transcriptase at the end of retrotranscription, both the two 99 nt-long overlapping (+) strands contain two adjacent tracts of guanines. This study demonstrates that oligonucleotides containing these G-clusters form highly stable G-quadruplexes of various structures in vitro, whose formation was controlled by an easy and reversible protocol using sodium hydroxide. Among these sequences, a G'2 hairpin dimer was the most stable structure adopted by the 5'-tail of the (+) downstream strand. Since the two (+) strands of the HIV-1 central DNA flap hold these G-clusters, and based on the properties of reverse branch migration in DNA flaps, constructions using HIV-1 sequences were assembled to mimic small DNA flaps where the G-clusters are neighbors. G-quartets were successfully probed in such flaps. They were induced by potassium and by a dibenzophenanthroline derivative already known to stabilize them. Such results suggest some function(s) for G-quartets associated with a DNA flap in the HIV-1 pre-integration steps, and argue for their transient formation during the processing of G-rich DNA flaps at the time of replication and/or repair.