Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system.

Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity.

Hierarchy of RNA functional dynamics.

RNA dynamics play a fundamental role in many cellular functions. However, there is no general framework to describe these complex processes, which typically consist of many structural maneuvers that occur over timescales ranging from picoseconds to seconds. Here, we classify RNA dynamics into distinct modes representing transitions between basins on a hierarchical free-energy landscape. These transitions include large-scale secondary-structural transitions at >0.1-s timescales, base-pair/tertiary dynamics at microsecond-to-millisecond timescales, stacking dynamics at timescales ranging from nanoseconds to microseconds, and other "jittering" motions at timescales ranging from picoseconds to nanoseconds. We review various modes within these three different tiers, the different mechanisms by which they are used to regulate function, and how they can be coupled together to achieve greater functional complexity.

Conservation and selective pressures shaping baleen whale olfactory receptor genes supports their use of olfaction in the marine environment.

The relative importance of various sensory modalities can shift in response to evolutionary transitions, resulting in changes to underlying gene families encoding their reception systems. The rapid birth-and-death process underlying the evolution of the large olfactory receptor (OR) gene family has accelerated genomic-level change for the sense of smell in particular. The transition from the land to sea in marine mammals is an attractive model for understanding the influence of habitat shifts on sensory systems, with the retained OR repertoire of baleen whales contrasting with its loss in toothed whales. In this study, we examine to what extent the transition from a terrestrial to a marine environment has influenced the evolution of baleen whale OR repertoires. We developed Gene Mining Pipeline (GMPipe) (https://github.com/AprilJauhal/GMPipe), which can accurately identify large numbers of candidate OR genes. GMPipe identified 707 OR sequences from eight baleen whale species. These repertoires exhibited distinct family count distributions compared to terrestrial mammals, including signs of relative expansion in families OR10, OR11 and OR13. While many receptors have been lost or show signs of random drift in baleen whales, others exhibit signs of evolving under purifying or positive selection. Over 85% of OR genes could be sorted into orthologous groups of sequences containing at least four homologous sequences. Many of these groups, particularly from family OR10, presented signs of relative expansion and purifying selective pressure. Overall, our results suggest that the relatively small size of baleen whale OR repertoires result from specialisation to novel olfactory landscapes, as opposed to random drift.

Identification of a novel gene PpBCG1 that has a positive role in desiccation tolerance in the moss Physcomitrium patens.

Water-to-land transition is a hallmark of terrestrialization for land plants and requires molecular adaptation to resist water deficiency. Lineages- or species-specific genes are widespread across eukaryotes, and yet the majority of those are functionally unknown and not annotated. Recent studies have revealed that some of such genes could play a role in adapting to environmental stress responses. Here, we identified a novel gene PpBCG1 (Bryophyte Co-retained Gene 1) in the moss Physcomitrium patens that was responsive to dehydration and rehydration. Under de- and rehydration treatments, PpBCG1 was significantly co-expressed with the dehydrin-encoding gene PpDHNA. Microarray data revealed that PpBCG1 was highly expressed in tissues of spores, female organ archegonia, and mature sporophytes. In addition, the Ppbcg1 mutant showed reduced ability of dehydration tolerance, whose plants were accompanied by a relatively low level of chlorophyll content during recovery. Comprehensive transcriptomics uncovered a detailed set of regulatory processes that were affected by the PpBCG1 disruption. Moreover, experimental evidence showed that PpBCG1 might function in the antioxidant activity, abscisic acid (ABA) pathway, and intracellular calcium (Ca2+) homeostasis to resist desiccation. Together, our study provides insights into the roles of one bryophyte co-retained gene in the desiccation tolerance.

Hydrothermally Stimulated Molecular Interfaces for Augmented Electron Delocalization in Wet-Chemical Phosphorus Recovery from Incineration Ash of Sewage Sludge.

Wet-chemically recovering phosphorus (P) from sewage sludge incineration ash (SSIA) has already become a global initiative to address P deficit, but effectively isolating P from these accompanying metals (AMs) through adsorption in a SSIA-derived extract remains elusive. Here, we devised a hydrothermal stimulus-motivated thermodynamic and kinetic enhancement to gain anionic ethylenediaminetetraacetic acid (EDTA) molecular interfaces for AM enclosure to resolve this conundrum. A new dosage rule based on the EDTA coordination ratio with AMs was established for the first time. Upon hydrothermal extraction at 140 °C for 1 h, the P extraction efficiency reached 96.7% or higher for these obtained SSIA samples, and then exceptional P sequestration from these EDTA-chelated AMs was realized by the peculiar lanthanum (La)-based nanoadsorbent (having 188.86 mg P/g adsorbent at pH ∼ 3.0). Relevant theoretical calculations unraveled that these delocalized electrons of tetravalent EDTA molecules boosted the enclosure of liberated AMs, thereby entailing a substantially increased negative adsorption energy (-408.7 kcal/mol) of P in the form of H2PO4- through intruding lattice-edged carbonates to coordinate La with monodentate mononuclear over LaCO5(1 0 1). This work highlights the prospect of molecular adaptation of these common extractants in wet-chemical P recovery from various P-included wastes, further sustaining global P circularity.

Analysis of selection in protein-coding sequences accounting for common biases.

The evolution of protein-coding genes is usually driven by selective processes, which favor some evolutionary trajectories over others, optimizing the subsequent protein stability and activity. The analysis of selection in this type of genetic data is broadly performed with the metric nonsynonymous/synonymous substitution rate ratio (dN/dS). However, most of the well-established methodologies to estimate this metric make crucial assumptions, such as lack of recombination or invariable codon frequencies along genes, which can bias the estimation. Here, we review the most relevant biases in the dN/dS estimation and provide a detailed guide to estimate this metric using state-of-the-art procedures that account for such biases, along with illustrative practical examples and recommendations. We also discuss the traditional interpretation of the estimated dN/dS emphasizing the importance of considering complementary biological information such as the role of the observed substitutions on the stability and function of proteins. This review is oriented to help evolutionary biologists that aim to accurately estimate selection in protein-coding sequences.

Coevolutionary escalation led to differentially adapted paralogs of an insect's Na,K-ATPase optimizing resistance to host plant toxins.

Cardiac glycosides are chemical defence toxins known to fatally inhibit the Na,K-ATPase (NKA) throughout the animal kingdom. Several animals, however, have evolved target-site insensitivity through substitutions in the otherwise highly conserved cardiac glycoside binding pocket of the NKA. The large milkweed bug, Oncopeltus fasciatus, shares a long evolutionary history with cardiac glycoside containing plants that led to intricate adaptations. Most strikingly, several duplications of the bugs' NKA1α gene provided the opportunity for differential resistance-conferring substitutions and subsequent sub-functionalization of the enzymes. Here, we analysed cardiac glycoside resistance and ion pumping activity of nine functional NKA α/ß-combinations of O. fasciatus expressed in cell culture. We tested the enzymes with two structurally distinct cardiac glycosides, calotropin, a host plant compound, and ouabain, a standard cardiac glycoside. The identity and number of known resistance-conferring substitutions in the cardiac glycoside binding site significantly impacted activity and toxin resistance in the three α-subunits. The ß-subunits also influenced the enzymes' characteristics, yet to a lesser extent. Enzymes containing the more ancient αC-subunit were inhibited by both compounds but much more strongly by the host plant toxin calotropin than by ouabain. The sensitivity to calotropin was diminished in enzymes containing the more derived αB and αA, which were only marginally inhibited by both cardiac glycosides. This trend culminated in αAß1 having higher resistance against calotropin than against ouabain. These results support the coevolutionary escalation of plant defences and herbivore tolerance mechanisms. The possession of multiple paralogs additionally mitigates pleiotropic effects by compromising between ion pumping activity and resistance.

Divergent selection and climate adaptation fuel genomic differentiation between sister species of Sphagnum (peat moss).

BACKGROUND AND AIMS: New plant species can evolve through the reinforcement of reproductive isolation via local adaptation along habitat gradients. Peat mosses (Sphagnaceae) are an emerging model system for the study of evolutionary genomics and have well-documented niche differentiation among species. Recent molecular studies have demonstrated that the globally distributed species Sphagnum magellanicum is a complex of morphologically cryptic lineages that are phylogenetically and ecologically distinct. Here, we describe the architecture of genomic differentiation between two sister species in this complex known from eastern North America: the northern S. diabolicum and the largely southern S. magniae. METHODS: We sampled plant populations from across a latitudinal gradient in eastern North America and performed whole genome and restriction-site associated DNA sequencing. These sequencing data were then analyzed computationally. KEY RESULTS: Using sliding-window population genetic analyses we find that differentiation is concentrated within 'islands' of the genome spanning up to 400 kb that are characterized by elevated genetic divergence, suppressed recombination, reduced nucleotide diversity and increased rates of non-synonymous substitution. Sequence variants that are significantly associated with genetic structure and bioclimatic variables occur within genes that have functional enrichment for biological processes including abiotic stress response, photoperiodism and hormone-mediated signalling. Demographic modelling demonstrates that these two species diverged no more than 225 000 generations ago with secondary contact occurring where their ranges overlap. CONCLUSIONS: We suggest that this heterogeneity of genomic differentiation is a result of linked selection and reflects the role of local adaptation to contrasting climatic zones in driving speciation. This research provides insight into the process of speciation in a group of ecologically important plants and strengthens our predictive understanding of how plant populations will respond as Earth's climate rapidly changes.

Heat stress adaptation in cows - Physiological responses and underlying molecular mechanisms.

Heat stress is a key abiotic stressor for dairy production in the tropics which is further compounded by the ongoing climate change. Heat stress not only adversely impacts the production and welfare of dairy cows but severely impacts the economics of dairying due to production losses and increased cost of rearing. Over the years, selection has ensured development of high producing breeds, however, the thermotolerance ability of animals has been largely overlooked. In the past decade, the ill effects of climate change have made it pertinent to rethink the selection strategies to opt for climate resilient breeds, to ensure optimum production and reproduction. This has led to renewed interest in evaluation of the impacts of heat stress on cows and the underlying mechanisms that results in their acclimatization and adaptation to varied thermal ambience. The understanding of heat stress and associated responses at various level of animal is crucial to device amelioration strategies to secure optimum production and welfare of cows. With this review, an effort has been made to provide an overview on temperature humidity index as an important indicator of heat stress, general effect of heat stress in dairy cows, and impact of heat stress and subsequent response at physiological, haematological, molecular and genetic level of dairy cows.

LTR Retroelements and Bird Adaptation to Arid Environments.

TEs are known to be among the main drivers in genome evolution, leading to the generation of evolutionary advantages that favor the success of organisms. The aim of this work was to investigate the TE landscape in bird genomes to look for a possible relationship between the amount of specific TE types and environmental changes that characterized the Oligocene era in Australia. Therefore, the mobilome of 29 bird species, belonging to a total of 11 orders, was analyzed. Our results confirmed that LINE retroelements are not predominant in all species of this evolutionary lineage and highlighted an LTR retroelement dominance in species with an Australian-related evolutionary history. The bird LTR retroelement expansion might have happened in response to the Earth's dramatic climate changes that occurred about 30 Mya, followed by a progressive aridification across most of Australian landmasses. Therefore, in birds, LTR retroelement burst might have represented an evolutionary advantage in the adaptation to arid/drought environments.

Dietary Diversification and Specialization in Neotropical Bats Facilitated by Early Molecular Evolution.

Dietary adaptation is a major feature of phenotypic and ecological diversification, yet the genetic basis of dietary shifts is poorly understood. Among mammals, Neotropical leaf-nosed bats (family Phyllostomidae) show unmatched diversity in diet; from a putative insectivorous ancestor, phyllostomids have radiated to specialize on diverse food sources including blood, nectar, and fruit. To assess whether dietary diversification in this group was accompanied by molecular adaptations for changing metabolic demands, we sequenced 89 transcriptomes across 58 species and combined these with published data to compare ∼13,000 protein coding genes across 66 species. We tested for positive selection on focal lineages, including those inferred to have undergone dietary shifts. Unexpectedly, we found a broad signature of positive selection in the ancestral phyllostomid branch, spanning genes implicated in the metabolism of all major macronutrients, yet few positively selected genes at the inferred switch to plantivory. Branches corresponding to blood- and nectar-based diets showed selection in loci underpinning nitrogenous waste excretion and glycolysis, respectively. Intriguingly, patterns of selection in metabolism genes were mirrored by those in loci implicated in craniofacial remodeling, a trait previously linked to phyllostomid dietary specialization. Finally, we show that the null model of the widely-used branch-site test is likely to be misspecified, with the implication that the test is too conservative and probably under-reports true cases of positive selection. Our findings point to a complex picture of adaptive radiation, in which the evolution of new dietary specializations has been facilitated by early adaptations combined with the generation of new genetic variation.

Vexitoxins: conotoxin-like venom peptides from predatory gastropods of the genus Vexillum.

Venoms of predatory marine cone snails are intensely studied because of the biomedical applications of the neuropeptides that they contain, termed conotoxins. Meanwhile some gastropod lineages have independently acquired secretory glands strikingly similar to the venom gland of cone snails, suggesting that they possess similar venoms. Here we focus on the most diversified of these clades, the genus Vexillum. Based on the analysis of a multi-species proteo-transcriptomic dataset, we show that Vexillum species indeed produce complex venoms dominated by highly diversified short cysteine-rich peptides, vexitoxins. Vexitoxins possess the same precursor organization, display overlapping cysteine frameworks and share several common post-translational modifications with conotoxins. Some vexitoxins show sequence similarity to conotoxins and adopt similar domain conformations, including a pharmacologically relevant inhibitory cysteine knot motif. The Vexillum envenomation gland (gL) is a notably more recent evolutionary novelty than the conoidean venom gland. Thus, we hypothesize lower divergence between vexitoxin genes, and their ancestral 'somatic' counterparts compared to that in conotoxins, and we find support for this hypothesis in the evolution of the vexitoxin cluster V027. We use this example to discuss how future studies on vexitoxins can inform the origin of conotoxins, and how they may help to address outstanding questions in venom evolution.

Molecular mechanisms underlying plasticity in a thermally varying environment.

Adaptation to environmental variability is a prerequisite for species' persistence in their natural environments. With climate change predicted to increase the frequency and severity of temperature fluctuations, ectothermic organisms may increasingly depend on acclimation capacity to accommodate thermal variability. To elucidate the molecular basis of fluctuating temperature-induced phenotypic plasticity, we investigated heat tolerance and the mechanisms induced by acclimation to thermal variability as compared to those seen at constant temperature. We ran genome-wide transcriptomic analysis on Drosophila melanogaster subjected to acclimation at constant (19 ± 0°C) and fluctuating (19 ± 8°C) temperatures and contrasted the induction of molecular mechanisms in adult males, adult females and larvae. We found life stage- and sex-specific dynamics of the acclimation responses to fluctuating temperatures. Adult flies exposed to temperature fluctuations showed a constitutive improvement in heat tolerance while heat tolerance of larvae tracked thermal fluctuations. A constitutive down-regulation of gene expression was observed for several genes in the larvae exposed to fluctuations. Our results for adult females showed that, for several genes, fluctuating temperature acclimation resulted in canalization of gene expression. Both transcriptional and post-transcriptional machinery were greatly affected by fluctuations in adult males. Gene ontology analysis showed enrichment of the heat stress response involving several major heat shock proteins in both larvae and adults exposed to fluctuating temperatures, even though fluctuations were in a benign range of temperatures. Finally, molecular mechanisms related to environmental sensing seem to be an important component of insect responses to thermal variability.

Effects of thermal acclimation on the proteome of the planarian Crenobia alpina from an alpine freshwater spring.

Species' acclimation capacity and their ability to maintain molecular homeostasis outside ideal temperature ranges will partly predict their success following climate change-induced thermal regime shifts. Theory predicts that ectothermic organisms from thermally stable environments have muted plasticity, and that these species may be particularly vulnerable to temperature increases. Whether such species retained or lost acclimation capacity remains largely unknown. We studied proteome changes in the planarian Crenobia alpina, a prominent member of cold-stable alpine habitats that is considered to be a cold-adapted stenotherm. We found that the species' critical thermal maximum (CTmax) is above its experienced habitat temperatures and that different populations exhibit differential CTmax acclimation capacity, whereby an alpine population showed reduced plasticity. In a separate experiment, we acclimated C. alpina individuals from the alpine population to 8, 11, 14 or 17°C over the course of 168 h and compared their comprehensively annotated proteomes. Network analyses of 3399 proteins and protein set enrichment showed that while the species' proteome is overall stable across these temperatures, protein sets functioning in oxidative stress response, mitochondria, protein synthesis and turnover are lower in abundance following warm acclimation. Proteins associated with an unfolded protein response, ciliogenesis, tissue damage repair, development and the innate immune system were higher in abundance following warm acclimation. Our findings suggest that this species has not suffered DNA decay (e.g. loss of heat-shock proteins) during evolution in a cold-stable environment and has retained plasticity in response to elevated temperatures, challenging the notion that stable environments necessarily result in muted plasticity.

Extended interaction networks with HCV protease NS3-4A substrates explain the lack of adaptive capability against protease inhibitors.

Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168 To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.

Lowered sensitivity of bitter taste receptors to ß-glucosides in bamboo lemurs: an instance of parallel and adaptive functional decline in TAS2R16?

Bitter taste facilitates the detection of potentially harmful substances and is perceived via bitter taste receptors (TAS2Rs) expressed on the tongue and oral cavity in vertebrates. In primates, TAS2R16 specifically recognizes ß-glucosides, which are important in cyanogenic plants' use of cyanide as a feeding deterrent. In this study, we performed cell-based functional assays for investigating the sensitivity of TAS2R16 to ß-glucosides in three species of bamboo lemurs (Prolemur simus, Hapalemur aureus and H. griseus), which primarily consume high-cyanide bamboo. TAS2R16 receptors from bamboo lemurs had lower sensitivity to ß-glucosides, including cyanogenic glucosides, than that of the closely related ring-tailed lemur (Lemur catta). Ancestral reconstructions of TAS2R16 for the bamboo-lemur last common ancestor (LCA) and that of the Hapalemur LCA showed an intermediate sensitivity to ß-glucosides between that of the ring-tailed lemurs and bamboo lemurs. Mutagenetic analyses revealed that P. simus and H. griseus had separate species-specific substitutions that led to reduced sensitivity. These results indicate that low sensitivity to ß-glucosides at the cellular level-a potentially adaptive trait for feeding on cyanogenic bamboo-evolved independently after the Prolemur-Hapalemur split in each species.

Genomic insights into molecular adaptation to intertidal environments in the mangrove Aegiceras corniculatum.

Mangroves have colonised extreme intertidal environments characterised by high salinity, hypoxia and other abiotic stresses. Aegiceras corniculatum, a pioneer mangrove species that has evolved two specialised adaptive traits (salt secretion and crypto-vivipary) is an attractive ecological model to investigate molecular mechanisms underlying adaptation to intertidal environments. We assembled de novo a high-quality reference genome of A. corniculatum and performed comparative genomic and transcriptomic analyses to investigate molecular mechanisms underlying adaptation to intertidal environments. We provide evidence that A. corniculatum experienced a whole-genome duplication (WGD) event c. 35 Ma. We infer that maintenance of cellular environmental homeostasis is an important adaptive process in A. corniculatum. The 14-3-3 and H+ -ATPase protein-coding genes, essential for the salt homeostasis, were preferentially retained after the recent WGD event. Using comparative transcriptomics, we show that genes upregulated under high-salt conditions are involved in salt transport and ROS scavenging. We also found that all homologues of DELAY OF GERMINATION1 (DOG1) had lost their heme-binding ability in A. corniculatum, and that this may contribute to crypto-vivipary. Our study provides insight into the genomic correlates of phenotypic adaptation to intertidal environments. This could contribute not only within the genomics community, but also to the field of plant evolution.

Molecular adaptations to heat stress in the thermophilic ant genus Cataglyphis.

Over the last decade, increasing attention has been paid to the molecular adaptations used by organisms to cope with thermal stress. However, to date, few studies have focused on thermophilic species living in hot, arid climates. In this study, we explored molecular adaptations to heat stress in the thermophilic ant genus Cataglyphis, one of the world's most thermotolerant animal taxa. We compared heat tolerance and gene expression patterns across six Cataglyphis species from distinct phylogenetic groups that live in different habitats and experience different thermal regimes. We found that all six species had high heat tolerance levels with critical thermal maxima (CTmax ) ranging from 43℃ to 45℃ and a median lethal temperature (LT50) ranging from 44.5℃ to 46.8℃. Transcriptome analyses revealed that, although the number of differentially expressed genes varied widely for the six species (from 54 to 1118), many were also shared. Functional annotation of the differentially expressed and co-expressed genes showed that the biological pathways involved in heat-shock responses were similar among species and were associated with four major processes: the regulation of transcriptional machinery and DNA metabolism; the preservation of proteome stability; the elimination of toxic residues; and the maintenance of cellular integrity. Overall, our results suggest that molecular responses to heat stress have been evolutionarily conserved in the ant genus Cataglyphis and that their diversity may help workers withstand temperatures close to their physiological limits.

Avian Coloration Genetics: Recent Advances and Emerging Questions.

The colorful phenotypes of birds have long provided rich source material for evolutionary biologists. Avian plumage, beaks, skin, and eggs-which exhibit a stunning range of cryptic and conspicuous forms-inspired early work on adaptive coloration. More recently, avian color has fueled discoveries on the physiological, developmental, and-increasingly-genetic mechanisms responsible for phenotypic variation. The relative ease with which avian color traits can be quantified has made birds an attractive system for uncovering links between phenotype and genotype. Accordingly, the field of avian coloration genetics is burgeoning. In this review, we highlight recent advances and emerging questions associated with the genetic underpinnings of bird color. We start by describing breakthroughs related to 2 pigment classes: carotenoids that produce red, yellow, and orange in most birds and psittacofulvins that produce similar colors in parrots. We then discuss structural colors, which are produced by the interaction of light with nanoscale materials and greatly extend the plumage palette. Structural color genetics remain understudied-but this paradigm is changing. We next explore how colors that arise from interactions among pigmentary and structural mechanisms may be controlled by genes that are co-expressed or co-regulated. We also identify opportunities to investigate genes mediating within-feather micropatterning and the coloration of bare parts and eggs. We conclude by spotlighting 2 research areas-mechanistic links between color vision and color production, and speciation-that have been invigorated by genetic insights, a trend likely to continue as new genomic approaches are applied to non-model species.

Protein Evolution in the Flaviviruses.

Proteins are commonly used as molecular targets against pathogens such as viruses and bacteria. However, pathogens can evolve rapidly permitting their populations to increase in protein diversity over time and thus escape to the activity of a molecular therapy. Subsequently, in order to design more durable and robust therapies as well as to understand viral evolution in a host and subsequent transmission, it is central to understand the evolution of pathogen proteins. This understanding can enable the detection of protein regions that can be potential targets for therapies and predict the emergence of molecular resistance against therapies. In this direction, two articles published recently in the Journal of Molecular Evolution investigated the evolution of proteomes of diverse flaviviruses, including Zika virus, Dengue virus and West Nile virus. Here I discuss the importance of considering the evolution of viral proteins, with the use of as realistic as possible models and methods that mimic protein evolution, to improve the design of antiviral therapies.