Major G-Quadruplex Form of HIV-1 LTR Reveals a (3 + 1) Folding Topology Containing a Stem-Loop

Nucleic acids can form noncanonical four-stranded structures called G-quadruplexes. G-quadruplex-forming sequences are found in several genomes including human and viruses. Previous studies showed that the G-rich sequence located in the U3 promoter region of the HIV-1 long terminal repeat (LTR) folds into a set of dynamically interchangeable G-quadruplex structures. G-quadruplexes formed in the LTR could act as silencer elements to regulate viral transcription. Stabilization of LTR G-quadruplexes by G-quadruplex-specific ligands resulted in decreased viral production, suggesting the possibility of targeting viral G-quadruplex structures for antiviral purposes. Among all the G-quadruplexes formed in the LTR sequence, LTR-III was shown to be the major G-quadruplex conformation in vitro. Here we report the NMR structure of LTR-III in K+ solution, revealing the formation of a unique quadruplex-duplex hybrid consisting of a three-layer (3 + 1) G-quadruplex scaffold, a 12-nt diagonal loop containing a conserved duplex-stem, a 3-nt lateral loop, a 1-nt propeller loop, and a V-shaped loop. Our structure showed several distinct features including a quadruplex-duplex junction, representing an attractive motif for drug targeting. The structure solved in this study may be used as a promising target to selectively impair the viral cycle

A Fragment-Based Approach for the Development of G-Quadruplex Ligands: Role of the Amidoxime Moiety

G-quadruplex (G4) nucleic acid structures have been reported to be involved in several human pathologies, including cancer, neurodegenerative disorders and infectious diseases; however, G4 targeting compounds still need implementation in terms of drug-like properties and selectivity in order to reach the clinical use. So far, G4 ligands have been mainly identified through high-throughput screening methods or design of molecules with pre-set features. Here, we describe the development of new heterocyclic ligands through a fragment-based drug discovery (FBDD) approach. The ligands were designed against the major G4 present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), the stabilization of which has been shown to suppress viral gene expression and replication. Our method is based on the generation of molecular fragment small libraries, screened against the target to further elaborate them into lead compounds. We screened 150 small molecules, composed by structurally and chemically different fragments, selected from commercially available and in-house compounds; synthetic elaboration yielded several G4 ligands and two final G4 binders, both embedding an amidoxime moiety; one of these two compounds showed preferential binding for the HIV-1 LTR G4. This work presents the discovery of a novel potential pharmacophore and highlights the possibility to apply a fragment-based approach to develop G4 ligands with unexpected chemical features

Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy

Leaders-cheaters in male group cooperation: differences in nonverbal communication and genetic factors

Here we report on the results of an experimental study investigating "who?" emerges as a leader in the context of male group cooperation and "how?" they do that. The study was designed based on the iterated Public Goods Game, played face-to-face in groups composed of four male strangers. The game involved interactions both with and without communication to allow the assessment of individual cooperative strategies, leadership potential, and individual features of positive nonverbal expressiveness during interactions. Along with the individual behavioural characteristics we have addressed personality traits (the Big Five) and an oxytocin receptor gene polymorphism (OXTR: SNP rs53576; A/G) as putative markers of individual sociability. Our results revealed that emergent leaders most often employed the strategy of unconditional cooperation ("altruism") and were characterized by enhanced positive facial expressiveness and extraversion compared to non-leaders. However, a fraction of emergent leaders (25%) turned out to be occasional free-riders ("cheaters"). Their distinctive features were the highest scores on extraversion, exaggerated activity in negotiations, and over-expression of positive nonverbal elements. Given the high efficiency of leaders-cheaters' behaviour, we consider this result as the evidence for supernormal stimuli functioning in humans. Moreover, leaders-cheaters were characterized by a specific allelic frequency of OXTR rs53576 (heterozygosity: AG). The homozygous GG variant of this SNP is argued to be associated with prosociality, and the AA, on the contrary, with poor sociability. The heterozygous variant (AG) probably is a compromise that enables its carriers to successfully combine high social skills with anti-social behavior (free-riding). This finding supports existing evidence on the role of OXTR rs53576 in human social behaviour

Synthesis, Binding and Antiviral Properties of Potent Core-Extended Naphthalene Diimides Targeting the HIV-1 Long Terminal Repeat Promoter G-Quadruplexes

We have previously reported that stabilization of the G-quadruplex structures in the HIV-1 long terminal repeat (LTR) promoter suppresses viral transcription. Here we sought to develop new G-quadruplex ligands to be exploited as antiviral compounds by enhancing binding toward the viral G-quadruplex structures. We synthesized naphthalene diimide derivatives with a lateral expansion of the aromatic core. The new compounds were able to bind/stabilize the G-quadruplex to a high extent, and some of them displayed clear-cut selectivity toward the viral G-quadruplexes with respect to the human telomeric G-quadruplexes. This feature translated into low nanomolar anti-HIV-1 activity toward two viral strains and encouraging selectivity indexes. The selectivity depended on specific recognition of LTR loop residues; the mechanism of action was ascribed to inhibition of LTR promoter activity in cells. This is the first example of G-quadruplex ligands that show increased selectivity toward the viral G-quadruplexes and display remarkable antiviral activity

HIV-1 Nucleocapsid Protein Unfolds Stable RNA G-Quadruplexes in the Viral Genome and Is Inhibited by G-Quadruplex Ligands

The G-quadruplexes that form in the HIV-1 RNA genome hinder progression of reverse transcriptase in vitro, but not in infected cells. We investigated the possibility that the HIV-1 nucleocapsid protein NCp7, which remains associated with the viral RNA during reverse transcription, modulated HIV-1 RNA G-quadruplex stability. By electrophoresis, circular dichroism, mass spectrometry, and reverse transcriptase stop assays, we demonstrated that NCp7 binds and unfolds the HIV-1 RNA G-quadruplexes and promotes DNA/RNA duplex formation, allowing reverse transcription to proceed. The G-quadruplex ligand BRACO-19 was able to partially counteract this effect. These results indicate NCp7 as the first known viral protein able to unfold RNA G-quadruplexes, and they explain how the extra-stable HIV-1 RNA G-quadruplexes are processed; they also point out that the reverse transcription process is hindered by G-quadruplex ligands at both reverse transcriptase and NCp7 level. This information can lead to the development of more effective anti-HIV-1 drugs with a new mechanism of action

Sex differences in cooperativeness:An experiment with Buryats in Southern Siberia

We report on an experimental study that was set up to reveal differences in the tendencies of men and women to cooperate in same-sex interactions. Former studies on this subject were mostly conducted in industrialized modern societies. In contrast, we tested the cooperation tendency among Buryats, a people from Southern Siberia of Mongolian origin. All subjects participated in (1) one iterated Public Goods Game in a group of four individuals of the same sex and (2) four one-shot Prisoner's Dilemma games with different partners of the same sex. The interactions were in a face-to-face setting, but any intentional communication during the experiments was prohibited. We found that Buryat men were more cooperative than Buryat women in both types of same-sex interactions. In particular, the fraction of men employing a strategy of unconditional cooperation in the iterated Public Goods Game was much higher (36%) than the fraction of unconditional cooperators among women (21%). In general, the behavior of men was less context dependent than the behavior of women. In both sexes, individuals who were more cooperative in one type of game tended to be more cooperative in the other type of game. Although direct communication was prohibited, the interaction partners in the Prisoner's Dilemma games employed the same strategy much more frequently than expected by chance. We conclude that, even among strangers, the exchange of subtle signals is sufficient to coordinate strategic decisions

Unravelling the many facets of human cooperation in an experimental study

Humans readily cooperate, even with strangers and without prospects of reciprocation. Despite thousands of studies, this finding is not well understood. Most studies focussed on a single aspect of cooperation and were conducted under anonymous conditions. However, cooperation is a multi-faceted phenomenon, involving generosity, readiness to share, fairness, trust, trustworthiness, and willingness to take cooperative risks. Here, we report findings of an experiment where subjects had to make decisions in ten situations representing different aspects of cooperation, both under anonymous and ‘personalised’ conditions. In an anonymous setting, we found considerable individual variation in each decision situation, while individuals were consistent both within and across situations. Prosocial tendencies such as generosity, trust, and trustworthiness were positively correlated, constituting a ‘cooperativeness syndrome’, but the tendency to punish non-cooperative individuals is not part of this syndrome. In a personalised setting, information on the appearance of the interaction partner systematically affected cooperation-related behaviour. Subjects were more cooperative toward interaction partners whose facial photographs were judged ‘generous’, ‘trustworthy’, ‘not greedy’, ‘happy’, ‘attractive’, and ‘not angry’ by a separate panel. However, individuals eliciting more cooperation were not more cooperative themselves in our experiment. Our study shows that a multi-faceted approach can reveal general behavioural tendencies underlying cooperation, but it also uncovers new puzzling features of human cooperation

Effects of sex and sex-related facial traits on trust and trustworthiness: An experimental study

The ability to trust others, including strangers, is a prerequisite for human cooperation. Economically it is not rational to trust strangers, as trust can be easily exploited. Still, generally, the level of trust toward strangers is relatively high. Trust is closely related to trustworthiness: when trusting others, one expects them to reciprocate. Some individuals elicit more trust than others. Apparently, humans use subtle cues for judging the trustworthiness of their interaction partners. Here, we report on an experiment that investigates trust and trustworthiness in a population of 176 mainly Dutch students. The aims of our study were: (1) to investigate how the sex of interaction partners and their facial appearance (femininity/masculinity) affect the degree of trust and trustworthiness, compared to fully anonymous conditions; (2) to test whether individuals who elicit trust in their interaction partners are trustworthy themselves. Each subject of our experiment played five one-shot Trust Games: one with an anonymous interaction partner, and four “personalized” games after seeing a 20 s silent video of their interaction partner (twice same-sex, and twice opposite-sex). The degree of facial sexual dimorphism was investigated with geometric morphometrics based on full-face photographs. Our results revealed that, despite the already high level of trust in the anonymous setting, the personalization of interactions had a clear effect on behavior. Females elicited more trust in partners of both sexes. Interestingly, females with more feminine faces elicited less trust in both male and female partners, while males with more masculine facial shape were more trusted by females, but less trusted by males. Neither sex nor facial femininity/masculinity predicted trustworthiness. Our results demonstrate that (1) sex and sex-related facial traits of interaction partners have a clear effect on eliciting trust in strangers. However, (2) these cues are not reliable predictors of actual trustworthiness