Emergence of compensatory mutations reveal the importance of electrostatic interactions between HIV-1 integrase and genomic RNA

Independent of its catalytic activity, HIV-1 integrase (IN) enzyme regulates proper particle maturation by binding to and packaging the viral RNA genome (gRNA) inside the mature capsid lattice. Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit the binding of IN to the gRNA and cause the formation of non-infectious virions characterized by mislocalization of the viral ribonucleoprotein complexes between the translucent conical capsid lattice and the viral lipid envelope. To gain insight into the molecular nature of IN-gRNA interactions, we have isolated compensatory substitutions in the background of a class II IN (R269A/K273A) variant that directly inhibits IN binding to the gRNA. We found that additional D256N and D270N substitutions in the C-terminal domain (CTD) of IN restored its ability to bind gRNA and led to the formation of infectious particles with correctly matured morphology. Furthermore, reinstating the overall positive electrostatic potential of the CTD through individual D256R or D256K substitutions was sufficient to restore IN-RNA binding and infectivity for the R269A/K273A as well as the R262A/R263A class II IN mutants. The compensatory mutations did not impact functional IN oligomerization, suggesting that they directly contributed to IN binding to the gRNA. Interestingly, HIV-1 IN R269A/K273A, but not IN R262A/R263A, bearing compensatory mutations was more sensitive to ALLINIs providing key genetic evidence that specific IN residues required for RNA binding also influence ALLINI activity. Structural modeling provided further insight into the molecular nature of IN-gRNA interactions and ALLINI mechanism of action. Taken together, our findings highlight an essential role of IN-gRNA interactions for proper virion maturation and reveal the importance of electrostatic interactions between the IN CTD and the gRNA.

Harmful behaviour through plasmid transfer: a successful evolutionary strategy of bacteria harbouring conjugative plasmids

Conjugative plasmids are extrachromosomal mobile genetic elements pervasive among bacteria. Plasmids' acquisition often lowers cells' growth rate, so their ubiquity has been a matter of debate. Chromosomes occasionally mutate, rendering plasmids cost-free. However, these compensatory mutations typically take hundreds of generations to appear after plasmid arrival. By then, it could be too late to compete with fast-growing plasmid-free cells successfully. Moreover, arriving plasmids would have to wait hundreds of generations for compensatory mutations to appear in the chromosome of their new host. We hypothesize that plasmid-donor cells may use the plasmid as a ‘weapon’ to compete with plasmid-free cells, particularly in structured environments. Cells already adapted to plasmids may increase their inclusive fitness through plasmid transfer to impose a cost to nearby plasmid-free cells and increase the replication opportunities of nearby relatives. A mathematical model suggests conditions under which the proposed hypothesis works, and computer simulations tested the long-term plasmid maintenance. Our hypothesis explains the maintenance of conjugative plasmids not coding for beneficial genes. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Computational Compensatory Mutation Discovery Approach: Predicting a PARP1 Variant Rescue Mutation

The prediction of protein mutations that affect function may be exploited for multiple uses. In the context of disease variants, the prediction of compensatory mutations that reestablish functional phenotypes could aid in the development of genetic therapies. In this work, we present an integrated approach that combines coevolutionary analysis and molecular dynamics (MD) simulations to discover functional compensatory mutations. This approach is employed to investigate possible rescue mutations of a poly(ADP-ribose) polymerase 1 (PARP1) variant, PARP1 V762A, associated with lung cancer and follicular lymphoma. MD simulations show PARP1 V762A exhibits noticeable changes in structural and dynamical behavior compared with wild type PARP1. Our integrated approach predicts A755E as a possible compensatory mutation based on coevolutionary information, and molecular simulations indicate that the PARP1 A755E/V762A double mutant exhibits similar structural and dynamical behavior to WT PARP1. Our methodology can be broadly applied to a large number of systems where SNPs have been identified as connected to disease and can shed light on the biophysical effects of such changes as well as provide a way to discover potential mutants that could restore wild type-like functionality. This can in turn be further utilized in the design of molecular therapeutics that aim to mimic such compensatory effect.

The structural determinants of intra-protein compensatory substitutions

Compensating substitutions happen when one mutation is advantageously selected because it restores the loss of fitness induced by a previous deleterious mutation. How frequent such mutations occur in evolution and what is the structural and functional context permitting their emergence remain open questions. We built an atlas of intra-protein compensatory substitutions using a phylogenetic approach and a dataset of 1,630 bacterial protein families for which high-quality sequence alignments and experimentally derived protein structures were available. We identified more than 51,000 positions coevolving by the mean of predicted compensatory mutations. Using the evolutionary and structural properties of the analyzed positions, we demonstrate that compensatory mutations are scarce (typically only a few in the protein history) but widespread (the majority of proteins experienced at least one). Typical coevolving residues are evolving slowly, are located in the protein core outside secondary structure motifs, and are more often in contact than expected by chance, even after accounting for their evolutionary rate and solvent exposure. An exception to this general scheme are residues coevolving for charge compensation, which are evolving faster than non-coevolving sites, in contradiction with predictions from simple coevolutionary models, but similar to stem pairs in RNA. While sites with a significant pattern of coevolution by compensatory mutations are rare, the comparative analysis of hundreds of structures ultimately permits a better understanding of the link between the three-dimensional structure of a protein and its fitness landscape.

Evolutionary instability of collateral susceptibility networks in ciprofloxacin resistant clinical Escherichia coli strains

Collateral sensitivity and resistance occur when resistance development towards one antimicrobial either potentiates or deteriorates the effect of others, respectively. Previous reports on collateral effects on susceptibility focus on newly acquired resistance determinants and propose that novel treatment guidelines informed by collateral networks may reduce the evolution, selection and spread of antimicrobial resistance. In this study, we investigate the evolutionary stability of collateral networks in five ciprofloxacin resistant, clinical Escherichia coli strains. After 300 generations of experimental evolution without antimicrobials, we show complete fitness restoration in four of five genetic backgrounds and demonstrate evolutionary instability in collateral networks of newly acquired resistance determinants. We show that compensatory mutations reducing efflux expression is the main driver destabilizing initial collateral networks and identify rpoS as a putative target for compensatory evolution. Our results add another layer of complexity to future predictions and clinical application of collateral networks.

Plasmid fitness costs are caused by specific genetic conflicts enabling resolution by compensatory mutation

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid carriage are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or gene expression level. By combining the results of experimental evolution with genetics and transcriptomics, we show here that fitness costs of 2 divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid carriage. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by up-regulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained up-regulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer (HGT) due to their propensity for amelioration by single compensatory mutations, helping to explain why plasmids are so common in bacterial genomes.

Mitonuclear interactions and introgression genomics of macaque monkeys ( Macaca ) highlight the influence of behaviour on genome evolution

In most macaques, females are philopatric and males migrate from their natal ranges, which results in pronounced divergence of mitochondrial genomes within and among species. We therefore predicted that some nuclear genes would have to acquire compensatory mutations to preserve compatibility with diverged interaction partners from the mitochondria. We additionally expected that these sex-differences would have distinctive effects on gene flow in the X and autosomes. Using new genomic data from 29 individuals from eight species of Southeast Asian macaque, we identified evidence of natural selection associated with mitonuclear interactions, including extreme outliers of interspecies differentiation and metrics of positive selection, low intraspecies polymorphism and atypically long runs of homozygosity associated with nuclear-encoded genes that interact with mitochondria-encoded genes. In one individual with introgressed mitochondria, we detected a small but significant enrichment of autosomal introgression blocks from the source species of her mitochondria that contained genes which interact with mitochondria-encoded loci. Our analyses also demonstrate that sex-specific demography sculpts genetic exchange across multiple species boundaries. These findings show that behaviour can have profound but indirect effects on genome evolution by influencing how interacting components of different genomic compartments (mitochondria, the autosomes and the sex chromosomes) move through time and space.

Targeted destabilization of the HIV-1 gp120-gp41 interface leads to convergent evolution with mutations in the V1V2, HR1 and HR2 domains

The HIV-1 envelope glycoprotein (Env) trimer is responsible for viral entry into target cells and is the sole target of neutralizing antibodies. The Env protein is therefore the focus of HIV-1 vaccine design. Env consists of two non-covalently linked subunits (gp120 and gp41) that form a trimer of heterodimers and this 6-subunit complex is metastable and conformationally flexible. Several approaches have been pursued to stabilize the Env trimer for vaccine purposes, which include structure-based design, high-throughput screening and selection by mammalian cell display. Here, we employed directed virus evolution to improve Env trimer stability. Accordingly, we deliberately destabilized the Env gp120-gp41 interface by mutagenesis in the context of replicating HIV-1 LAI virus and virus evolution over time. We identified compensatory changes that pointed at convergent evolution as they were largely restricted to specific Env regions, namely the V1V2-domain of gp120, and the the HR1 and HR2 domain of gp41. Specifically, S614G in V1V2 and Q567R in HR1 were frequently identified. Interestingly, the majority of the compensatory mutations were at distant locations from the original mutations and most likely strengthen inter-subunit interactions. These results show how the virus can overcome Env instability and illuminate the regions that play a dominant role in Env stability. Importance A successful HIV-1 vaccine most likely requires an envelope glycoprotein (Env) component, as the Env is the only viral protein on the surface of the virus and the target for neutralizing antibodies. However, HIV Env is metastable and flexible because of the weak interactions between the Env subunits, complicating the generation of recombinant mimics of native Env. Here, we used directed viral evolution to study Env stability. We deliberately destabilized the interface between Env subunits and explored the capacity of the virus to repair trimer instability by evolution. We identified compensatory mutations that converged in specific Env locations: the apex and the trimer interface. Selected mutations enhanced the stability of recombinant soluble Env trimer proteins. These results provided clues on understanding the structural mechanisms involved in Env trimer stability, which can guide future immunogen design.

Saturation Mutagenesis of the Transmembrane Region of HokC in Escherichia coli Reveals Its High Tolerance to Mutations

Cells adapt to different stress conditions, such as the antibiotics presence. This adaptation sometimes is achieved by changing relevant protein positions, of which the mutability is limited by structural constrains. Understanding the basis of these constrains represent an important challenge for both basic science and potential biotechnological applications. To study these constraints, we performed a systematic saturation mutagenesis of the transmembrane region of HokC, a toxin used by Escherichia coli to control its own population, and observed that 92% of single-point mutations are tolerated and that all the non-tolerated mutations have compensatory mutations that reverse their effect. We provide experimental evidence that HokC accumulates multiple compensatory mutations that are found as correlated mutations in the HokC family multiple sequence alignment. In agreement with these observations, transmembrane proteins show higher probability to present correlated mutations and are less densely packed locally than globular proteins; previous mutagenesis results on transmembrane proteins further support our observations on the high tolerability to mutations of transmembrane regions of proteins. Thus, our experimental results reveal the HokC transmembrane region high tolerance to loss-of-function mutations that is associated with low sequence conservation and high rate of correlated mutations in the HokC family sequences alignment, which are features shared with other transmembrane proteins.

Derivation and characterization of an HIV-1 mutant that rescues IP6 binding deficiency

Background A critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation are facilitated by the cellular polyanion inositol hexaphosphate (IP6), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP6 binding deficiency, we passaged HIV-1 mutants that had substitutions in IP6 coordinating residues to select for compensatory mutations. Results We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP6-binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP6-binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image BVM-induced assembly of individual HIV-1 particles assembly in living cells. Conclusions Overall these results suggest that IP6-Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can rescue IP6 deficiency. Additionally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.