The Diverse Evolutionary Histories of Domesticated Metaviral Capsid Genes in Mammals.

Selfish genetic elements comprise significant fractions of mammalian genomes. In rare instances, host genomes domesticate segments of these elements for function. Using a complete human genome assembly and 25 additional vertebrate genomes, we re-analyzed the evolutionary trajectories and functional potential of capsid (CA) genes domesticated from Metaviridae, a lineage of retrovirus-like retrotransposons. Our study expands on previous analyses to unearth several new insights about the evolutionary histories of these ancient genes. We find that at least five independent domestication events occurred from diverse Metaviridae, giving rise to three universally retained single-copy genes evolving under purifying selection and two gene families unique to placental mammals, with multiple members showing evidence of rapid evolution. In the SIRH/RTL family, we find diverse amino-terminal domains, widespread loss of protein-coding capacity in RTL10 despite its retention in several mammalian lineages, and differential utilization of an ancient programmed ribosomal frameshift in RTL3 between the domesticated CA and protease domains. Our analyses also reveal that most members of the PNMA family in mammalian genomes encode a conserved putative amino-terminal RNA-binding domain (RBD) both adjoining and independent from domesticated CA domains. Our analyses lead to a significant correction of previous annotations of the essential CCDC8 gene. We show that this putative RBD is also present in several extant Metaviridae, revealing a novel protein domain configuration in retrotransposons. Collectively, our study reveals the divergent outcomes of multiple domestication events from diverse Metaviridae in the common ancestor of placental mammals.

The diverse evolutionary histories of domesticated metaviral capsid genes in mammals.

Selfish genetic elements and their remnants comprise at least half of the human genome. Active transposons duplicate by inserting copies at new sites in a host genome. Following insertion, transposons can acquire mutations that render them inactive; the accrual of additional mutations can render them unrecognizable over time. However, in rare instances, segments of transposons become useful for the host, in a process called gene domestication. Using the first complete human genome assembly and 25 additional vertebrate genomes, we analyzed the evolutionary trajectories and functional potential of genes domesticated from the capsid genes of Metaviridae, a retroviral-like retrotransposon family. Our analysis reveals four families of domesticated capsid genes in placental mammals with varied evolutionary outcomes, ranging from universal retention to lineage-specific duplications or losses and from purifying selection to lineage-specific rapid evolution. The four families of domesticated capsid genes have divergent amino-terminal domains, inherited from four distinct ancestral metaviruses. Structural predictions reveal that many domesticated genes encode a previously unrecognized RNA-binding domain retained in multiple paralogs in mammalian genomes both adjacent to and independent from the capsid domain. Collectively, our study reveals diverse outcomes of domestication of diverse metaviruses, which led to structurally and evolutionarily diverse genes that encode important, but still largely-unknown functions in placental mammals. (207).

A functional map of HIV-host interactions in primary human T cells.

Human Immunodeficiency Virus (HIV) relies on host molecular machinery for replication. Systematic attempts to genetically or biochemically define these host factors have yielded hundreds of candidates, but few have been functionally validated in primary cells. Here, we target 426 genes previously implicated in the HIV lifecycle through protein interaction studies for CRISPR-Cas9-mediated knock-out in primary human CD4+ T cells in order to systematically assess their functional roles in HIV replication. We achieve efficient knockout (>50% of alleles) in 364 of the targeted genes and identify 86 candidate host factors that alter HIV infection. 47 of these factors validate by multiplex gene editing in independent donors, including 23 factors with restrictive activity. Both gene editing efficiencies and HIV-1 phenotypes are highly concordant among independent donors. Importantly, over half of these factors have not been previously described to play a functional role in HIV replication, providing numerous novel avenues for understanding HIV biology. These data further suggest that host-pathogen protein-protein interaction datasets offer an enriched source of candidates for functional host factor discovery and provide an improved understanding of the mechanics of HIV replication in primary T cells.

Novel Classes and Evolutionary Turnover of Histone H2B Variants in the Mammalian Germline.

Histones and their posttranslational modifications facilitate diverse chromatin functions in eukaryotes. Core histones (H2A, H2B, H3, and H4) package genomes after DNA replication. In contrast, variant histones promote specialized chromatin functions, including DNA repair, genome stability, and epigenetic inheritance. Previous studies have identified only a few H2B variants in animals; their roles and evolutionary origins remain largely unknown. Here, using phylogenomic analyses, we reveal the presence of five H2B variants broadly present in mammalian genomes. Three of these variants have been previously described: H2B.1, H2B.L (also called subH2B), and H2B.W. In addition, we identify and describe two new variants: H2B.K and H2B.N. Four of these variants originated in mammals, whereas H2B.K arose prior to the last common ancestor of bony vertebrates. We find that though H2B variants are subject to high gene turnover, most are broadly retained in mammals, including humans. Despite an overall signature of purifying selection, H2B variants evolve more rapidly than core H2B with considerable divergence in sequence and length. All five H2B variants are expressed in the germline. H2B.K and H2B.N are predominantly expressed in oocytes, an atypical expression site for mammalian histone variants. Our findings suggest that H2B variants likely encode potentially redundant but vital functions via unusual chromatin packaging or nonchromatin functions in mammalian germline cells. Our discovery of novel histone variants highlights the advantages of comprehensive phylogenomic analyses and provides unique opportunities to study how innovations in chromatin function evolve.

An actin-related protein that is most highly expressed in Drosophila testes is critical for embryonic development.

Most actin-related proteins (Arps) are highly conserved and carry out well-defined cellular functions in eukaryotes. However, many lineages like Drosophila and mammals encode divergent non-canonical Arps whose roles remain unknown. To elucidate the function of non-canonical Arps, we focus on Arp53D, which is highly expressed in testes and retained throughout Drosophila evolution. We show that Arp53D localizes to fusomes and actin cones, two germline-specific actin structures critical for sperm maturation, via a unique N-terminal tail. Surprisingly, we find that male fertility is not impaired upon Arp53D loss, yet population cage experiments reveal that Arp53D is required for optimal fitness in Drosophila melanogaster. To reconcile these findings, we focus on Arp53D function in ovaries and embryos where it is only weakly expressed. We find that under heat stress Arp53D-knockout (KO) females lay embryos with reduced nuclear integrity and lower viability; these defects are further exacerbated in Arp53D-KO embryos. Thus, despite its relatively recent evolution and primarily testis-specific expression, non-canonical Arp53D is required for optimal embryonic development in Drosophila.

Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases.

RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.

Innovation of heterochromatin functions drives rapid evolution of essential ZAD-ZNF genes in Drosophila.

Contrary to dogma, evolutionarily young and dynamic genes can encode essential functions. We find that evolutionarily dynamic ZAD-ZNF genes, which encode the most abundant class of insect transcription factors, are more likely to encode essential functions in Drosophila melanogaster than ancient, conserved ZAD-ZNF genes. We focus on the Nicknack ZAD-ZNF gene, which is evolutionarily young, poorly retained in Drosophila species, and evolves under strong positive selection. Yet we find that it is necessary for larval development in D. melanogaster. We show that Nicknack encodes a heterochromatin-localizing protein like its paralog Oddjob, also an evolutionarily dynamic yet essential ZAD-ZNF gene. We find that the divergent D. simulans Nicknack protein can still localize to D. melanogaster heterochromatin and rescue viability of female but not male Nicknack-null D. melanogaster. Our findings suggest that innovation for rapidly changing heterochromatin functions might generally explain the essentiality of many evolutionarily dynamic ZAD-ZNF genes in insects.

A natural variant of the essential host gene MMS21 restricts the parasitic 2-micron plasmid in Saccharomyces cerevisiae.

Antagonistic coevolution with selfish genetic elements (SGEs) can drive evolution of host resistance. Here, we investigated host suppression of 2-micron (2µ) plasmids, multicopy nuclear parasites that have co-evolved with budding yeasts. We developed SCAMPR (Single-Cell Assay for Measuring Plasmid Retention) to measure copy number heterogeneity and 2µ plasmid loss in live cells. We identified three S. cerevisiae strains that lack endogenous 2µ plasmids and reproducibly inhibit mitotic plasmid stability. Focusing on the Y9 ragi strain, we determined that plasmid restriction is heritable and dominant. Using bulk segregant analysis, we identified a high-confidence Quantitative Trait Locus (QTL) with a single variant of MMS21 associated with increased 2µ instability. MMS21 encodes a SUMO E3 ligase and an essential component of the Smc5/6 complex, involved in sister chromatid cohesion, chromosome segregation, and DNA repair. Our analyses leverage natural variation to uncover a novel means by which budding yeasts can overcome highly successful genetic parasites.

Antiviral Activity and Adaptive Evolution of Avian Tetherins.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

Hybridization promotes asexual reproduction in Caenorhabditis nematodes.

Although most unicellular organisms reproduce asexually, most multicellular eukaryotes are obligately sexual. This implies that there are strong barriers that prevent the origin or maintenance of asexuality arising from an obligately sexual ancestor. By studying rare asexual animal species we can gain a better understanding of the circumstances that facilitate their evolution from a sexual ancestor. Of the known asexual animal species, many originated by hybridization between two ancestral sexual species. The balance hypothesis predicts that genetic incompatibilities between the divergent genomes in hybrids can modify meiosis and facilitate asexual reproduction, but there are few instances where this has been shown. Here we report that hybridizing two sexual Caenorhabditis nematode species (C. nouraguensis females and C. becei males) alters the normal inheritance of the maternal and paternal genomes during the formation of hybrid zygotes. Most offspring of this interspecies cross die during embryogenesis, exhibiting inheritance of a diploid C. nouraguensis maternal genome and incomplete inheritance of C. becei paternal DNA. However, a small fraction of offspring develop into viable adults that can be either fertile or sterile. Fertile offspring are produced asexually by sperm-dependent parthenogenesis (also called gynogenesis or pseudogamy); these progeny inherit a diploid maternal genome but fail to inherit a paternal genome. Sterile offspring are hybrids that inherit both a diploid maternal genome and a haploid paternal genome. Whole-genome sequencing of individual viable worms shows that diploid maternal inheritance in both fertile and sterile offspring results from an altered meiosis in C. nouraguensis oocytes and the inheritance of two randomly selected homologous chromatids. We hypothesize that hybrid incompatibility between C. nouraguensis and C. becei modifies maternal and paternal genome inheritance and indirectly induces gynogenetic reproduction. This system can be used to dissect the molecular mechanisms by which hybrid incompatibilities can facilitate the emergence of asexual reproduction.

Combinatorial mutagenesis of rapidly evolving residues yields super-restrictor antiviral proteins.

Antagonistic interactions drive host-virus evolutionary arms races, which often manifest as recurrent amino acid changes (i.e., positive selection) at their protein-protein interaction interfaces. Here, we investigated whether combinatorial mutagenesis of positions under positive selection in a host antiviral protein could enhance its restrictive properties. We tested approximately 700 variants of human MxA, generated by combinatorial mutagenesis, for their ability to restrict Thogotovirus (THOV). We identified MxA super-restrictors with increased binding to the THOV nucleoprotein (NP) target protein and 10-fold higher anti-THOV restriction relative to wild-type human MxA, the most potent naturally occurring anti-THOV restrictor identified. Our findings reveal a means to elicit super-restrictor antiviral proteins by leveraging signatures of positive selection. Although some MxA super-restrictors of THOV were impaired in their restriction of H5N1 influenza A virus (IAV), other super-restrictor variants increased THOV restriction without impairment of IAV restriction. Thus, broadly acting antiviral proteins such as MxA mitigate breadth-versus-specificity trade-offs that could otherwise constrain their adaptive landscape.

Killer Meiotic Drive and Dynamic Evolution of the wtf Gene Family.

Natural selection works best when the two alleles in a diploid organism are transmitted to offspring at equal frequencies. Despite this, selfish loci known as meiotic drivers that bias their own transmission into gametes are found throughout eukaryotes. Drive is thought to be a powerful evolutionary force, but empirical evolutionary analyses of drive systems are limited by low numbers of identified meiotic drive genes. Here, we analyze the evolution of the wtf gene family of Schizosaccharomyces pombe that contains both killer meiotic drive genes and suppressors of drive. We completed assemblies of all wtf genes for two S. pombe isolates, as well as a subset of wtf genes from over 50 isolates. We find that wtf copy number can vary greatly between isolates and that amino acid substitutions, expansions and contractions of DNA sequence repeats, and nonallelic gene conversion between family members all contribute to dynamic wtf gene evolution. This work demonstrates the power of meiotic drive to foster rapid evolution and identifies a recombination mechanism through which transposons can indirectly mobilize meiotic drivers.

Evolutionary origins and diversification of testis-specific short histone H2A variants in mammals.

Eukaryotic genomes must accomplish both compact packaging for genome stability and inheritance, as well as accessibility for gene expression. They do so using post-translational modifications of four ancient canonical histone proteins (H2A, H2B, H3, and H4) and by deploying histone variants with specialized chromatin functions. Some histone variants are conserved across all eukaryotes, whereas others are lineage-specific. Here, we performed detailed phylogenomic analyses of "short H2A histone" variants found in mammalian genomes. We discovered a previously undescribed typically-sized H2A variant in monotremes and marsupials, H2A.R, which may represent the common ancestor of the short H2As. We also discovered a novel class of short H2A histone variants in eutherian mammals, H2A.Q We show that short H2A variants arose on the X Chromosome in the common ancestor of all eutherian mammals and diverged into four evolutionarily distinct clades: H2A.B, H2A.L, H2A.P, and H2A.Q However, the repertoires of short histone H2A variants vary extensively among eutherian mammals due to lineage-specific gains and losses. Finally, we show that all four short H2As are subject to accelerated rates of protein evolution relative to both canonical and other variant H2A proteins including H2A.R. Our analyses reveal that short H2As are a unique class of testis-restricted histone variants displaying an unprecedented evolutionary dynamism. Based on their X-Chromosomal localization, genetic turnover, and testis-specific expression, we hypothesize that short H2A variants may participate in genetic conflicts involving sex chromosomes during reproduction.

wtf genes are prolific dual poison-antidote meiotic drivers.

Meiotic drivers are selfish genes that bias their transmission into gametes, defying Mendelian inheritance. Despite the significant impact of these genomic parasites on evolution and infertility, few meiotic drive loci have been identified or mechanistically characterized. Here, we demonstrate a complex landscape of meiotic drive genes on chromosome 3 of the fission yeasts Schizosaccharomyces kambucha and S. pombe. We identify S. kambucha wtf4 as one of these genes that acts to kill gametes (known as spores in yeast) that do not inherit the gene from heterozygotes. wtf4 utilizes dual, overlapping transcripts to encode both a gamete-killing poison and an antidote to the poison. To enact drive, all gametes are poisoned, whereas only those that inherit wtf4 are rescued by the antidote. Our work suggests that the wtf multigene family proliferated due to meiotic drive and highlights the power of selfish genes to shape genomes, even while imposing tremendous costs to fertility.

A mitochondrial DNA hypomorph of cytochrome oxidase specifically impairs male fertility in Drosophila melanogaster.

Due to their strict maternal inheritance in most animals and plants, mitochondrial genomes are predicted to accumulate mutations that are beneficial or neutral in females but harmful in males. Although a few male-harming mtDNA mutations have been identified, consistent with this 'Mother's Curse', their effect on females has been largely unexplored. Here, we identify COII(G177S), a mtDNA hypomorph of cytochrome oxidase II, which specifically impairs male fertility due to defects in sperm development and function without impairing other male or female functions. COII(G177S) represents one of the clearest examples of a 'male-harming' mtDNA mutation in animals and suggest that the hypomorphic mtDNA mutations like COII(G177S) might specifically impair male gametogenesis. Intriguingly, some D. melanogaster nuclear genetic backgrounds can fully rescue COII(G177S) -associated sterility, consistent with previously proposed models that nuclear genomes can regulate the phenotypic manifestation of mtDNA mutations.

Evolution of a transcriptional regulator from a transmembrane nucleoporin.

Nuclear pore complexes (NPCs) emerged as nuclear transport channels in eukaryotic cells ∼1.5 billion years ago. While the primary role of NPCs is to regulate nucleo-cytoplasmic transport, recent research suggests that certain NPC proteins have additionally acquired the role of affecting gene expression at the nuclear periphery and in the nucleoplasm in metazoans. Here we identify a widely expressed variant of the transmembrane nucleoporin (Nup) Pom121 (named sPom121, for "soluble Pom121") that arose by genomic rearrangement before the divergence of hominoids. sPom121 lacks the nuclear membrane-anchoring domain and thus does not localize to the NPC. Instead, sPom121 colocalizes and interacts with nucleoplasmic Nup98, a previously identified transcriptional regulator, at gene promoters to control transcription of its target genes in human cells. Interestingly, sPom121 transcripts appear independently in several mammalian species, suggesting convergent innovation of Nup-mediated transcription regulation during mammalian evolution. Our findings implicate alternate transcription initiation as a mechanism to increase the functional diversity of NPC components.

Differences in codon bias and GC content contribute to the balanced expression of TLR7 and TLR9.

The innate immune system detects diverse microbial species with a limited repertoire of immune receptors that recognize nucleic acids. The cost of this immune surveillance strategy is the potential for inappropriate recognition of self-derived nucleic acids and subsequent autoimmune disease. The relative expression of two closely related receptors, Toll-like receptor (TLR) 7 and TLR9, is balanced to allow recognition of microbial nucleic acids while limiting recognition of self-derived nucleic acids. Situations that tilt this balance toward TLR7 promote inappropriate responses, including autoimmunity; therefore, tight control of expression is critical for proper homeostasis. Here we report that differences in codon bias limit TLR7 expression relative to TLR9. Codon optimization of Tlr7 increases protein levels as well as responses to ligands, but, unexpectedly, these changes only modestly affect translation. Instead, we find that much of the benefit attributed to codon optimization is actually the result of enhanced transcription. Our findings, together with other recent examples, challenge the dogma that codon optimization primarily increases translation. We propose that suboptimal codon bias, which correlates with low guanine-cytosine (GC) content, limits transcription of certain genes. This mechanism may establish low levels of proteins whose overexpression leads to particularly deleterious effects, such as TLR7.

The piggyBac transposon-derived genes TPB1 and TPB6 mediate essential transposon-like excision during the developmental rearrangement of key genes in Tetrahymena thermophila.

Ciliated protozoans perform extreme forms of programmed somatic DNA rearrangement during development. The model ciliate Tetrahymena thermophila removes 34% of its germline micronuclear genome from somatic macronuclei by excising thousands of internal eliminated sequences (IESs), a process that shares features with transposon excision. Indeed, piggyBac transposon-derived genes are necessary for genome-wide IES excision in both Tetrahymena (TPB2 [Tetrahymena piggyBac-like 2] and LIA5) and Paramecium tetraurelia (PiggyMac). T. thermophila has at least three other piggyBac-derived genes: TPB1, TPB6, and TPB7 Here, we show that TPB1 and TPB6 excise a small, distinct set of 12 unusual IESs that disrupt exons. TPB1-deficient cells complete mating, but their progeny exhibit slow growth, giant vacuoles, and osmotic shock sensitivity due to retention of an IES in the vacuolar gene DOP1 (Dopey domain-containing protein). Unlike most IESs, TPB1-dependent IESs have piggyBac-like terminal inverted motifs that are necessary for excision. Transposon-like excision mediated by TPB1 and TPB6 provides direct evidence for a transposon origin of not only IES excision machinery but also IESs themselves. Our study highlights a division of labor among ciliate piggyBac-derived genes, which carry out mutually exclusive categories of excision events mediated by either transposon-like features or RNA-directed heterochromatin.

Evolutionary Analyses Suggest a Function of MxB Immunity Proteins Beyond Lentivirus Restriction.

Viruses impose diverse and dynamic challenges on host defenses. Diversifying selection of codons and gene copy number variation are two hallmarks of genetic innovation in antiviral genes engaged in host-virus genetic conflicts. The myxovirus resistance (Mx) genes encode interferon-inducible GTPases that constitute a major arm of the cell-autonomous defense against viral infection. Unlike the broad antiviral activity of MxA, primate MxB was recently shown to specifically inhibit lentiviruses including HIV-1. We carried out detailed evolutionary analyses to investigate whether genetic conflict with lentiviruses has shaped MxB evolution in primates. We found strong evidence for diversifying selection in the MxB N-terminal tail, which contains molecular determinants of MxB anti-lentivirus specificity. However, we found no overlap between previously-mapped residues that dictate lentiviral restriction and those that have evolved under diversifying selection. Instead, our findings are consistent with MxB having a long-standing and important role in the interferon response to viral infection against a broader range of pathogens than is currently appreciated. Despite its critical role in host innate immunity, we also uncovered multiple functional losses of MxB during mammalian evolution, either by pseudogenization or by gene conversion from MxA genes. Thus, although the majority of mammalian genomes encode two Mx genes, this apparent stasis masks the dramatic effects that recombination and diversifying selection have played in shaping the evolutionary history of Mx genes. Discrepancies between our study and previous publications highlight the need to account for recombination in analyses of positive selection, as well as the importance of using sequence datasets with appropriate depth of divergence. Our study also illustrates that evolutionary analyses of antiviral gene families are critical towards understanding molecular principles that govern host-virus interactions and species-specific susceptibility to viral infection.