RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions.

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.

Acquired stress resilience through bacteria-to-nematode interdomain horizontal gene transfer.

Natural selection drives the acquisition of organismal resilience traits to protect against adverse environments. Horizontal gene transfer (HGT) is an important evolutionary mechanism for the acquisition of novel traits, including metazoan acquisitions in immunity, metabolic, and reproduction function via interdomain HGT (iHGT) from bacteria. Here, we report that the nematode gene rml-3 has been acquired by iHGT from bacteria and that it enables exoskeleton resilience and protection against environmental toxins in Caenorhabditis elegans. Phylogenetic analysis reveals that diverse nematode RML-3 proteins form a single monophyletic clade most similar to bacterial enzymes that biosynthesize L-rhamnose, a cell-wall polysaccharide component. C. elegans rml-3 is highly expressed during larval development and upregulated in developing seam cells upon heat stress and during the stress-resistant dauer stage. rml-3 deficiency impairs cuticle integrity, barrier functions, and nematode stress resilience, phenotypes that can be rescued by exogenous L-rhamnose. We propose that interdomain HGT of an ancient bacterial rml-3 homolog has enabled L-rhamnose biosynthesis in nematodes, facilitating cuticle integrity and organismal resilience to environmental stressors during evolution. These findings highlight a remarkable contribution of iHGT on metazoan evolution conferred by the domestication of a bacterial gene.

Host-specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome.

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8's ability to sense coronavirus 3CLpros and, instead, enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intraspecies variation in inflammasome-mediated viral sensing and immunopathology.

Bacterial origin of a key innovation in the evolution of the vertebrate eye.

The vertebrate eye was described by Charles Darwin as one of the greatest potential challenges to a theory of natural selection by stepwise evolutionary processes. While numerous evolutionary transitions that led to the vertebrate eye have been explained, some aspects appear to be vertebrate specific with no obvious metazoan precursor. One critical difference between vertebrate and invertebrate vision hinges on interphotoreceptor retinoid-binding protein (IRBP, also known as retinol-binding protein, RBP3), which enables the physical separation and specialization of cells in the vertebrate visual cycle by promoting retinoid shuttling between cell types. While IRBP has been functionally described, its evolutionary origin has remained elusive. Here, we show that IRBP arose via acquisition of novel genetic material from bacteria by interdomain horizontal gene transfer (iHGT). We demonstrate that a gene encoding a bacterial peptidase was acquired prior to the radiation of extant vertebrates >500 Mya and underwent subsequent domain duplication and neofunctionalization to give rise to vertebrate IRBP. Our phylogenomic analyses on >900 high-quality genomes across the tree of life provided the resolution to distinguish contamination in genome assemblies from true instances of horizontal acquisition of IRBP and led us to discover additional independent transfers of the same bacterial peptidase gene family into distinct eukaryotic lineages. Importantly, this work illustrates the evolutionary basis of a key transition that led to the vertebrate visual cycle and highlights the striking impact that acquisition of bacterial genes has had on vertebrate evolution.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection.

Viral infection both activates stress signaling pathways and redistributes ribosomes away from host mRNAs to translate viral mRNAs. The intricacies of this ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome-associated quality control (RQC) factor ZNF598 during vaccinia virus mRNA translation. ZNF598 acts on collided ribosomes to ubiquitylate 40S subunit proteins uS10 (RPS20) and eS10 (RPS10), initiating RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection enhances uS10 ubiquitylation, indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells that either lack ZNF598 or express a ubiquitylation-deficient version of uS10. Using SILAC-based proteomics and concurrent RNA-seq analysis, we determine that translation, but not transcription of vaccinia virus mRNAs is compromised in cells with deficient RQC activity. Additionally, vaccinia virus infection reduces cellular RQC activity, suggesting that co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

Molecular basis for specific recognition of bacterial ligands by NAIP/NLRC4 inflammasomes.

NLR (nucleotide-binding domain [NBD]- and leucine-rich repeat [LRR]-containing) proteins mediate innate immune sensing of pathogens in mammals and plants. How NLRs detect their cognate stimuli remains poorly understood. Here, we analyzed ligand recognition by NLR apoptosis inhibitory protein (NAIP) inflammasomes. Mice express multiple highly related NAIP paralogs that recognize distinct bacterial proteins. We analyzed a panel of 43 chimeric NAIPs, allowing us to map the NAIP domain responsible for specific ligand detection. Surprisingly, ligand specificity was mediated not by the LRR domain, but by an internal region encompassing several NBD-associated α-helical domains. Interestingly, we find that the ligand specificity domain has evolved under positive selection in both rodents and primates. We further show that ligand binding is required for the subsequent co-oligomerization of NAIPs with the downstream signaling adaptor NLR family, CARD-containing 4 (NLRC4). These data provide a molecular basis for how NLRs detect ligands and assemble into inflammasomes.

Transferred interbacterial antagonism genes augment eukaryotic innate immune function.

Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.

Rules of engagement: molecular insights from host-virus arms races.

Mammalian genes and genomes have been shaped by ancient and ongoing challenges from viruses. These genetic imprints can be identified via evolutionary analyses to reveal fundamental details about when (how old), where (which protein domains), and how (what are the functional consequences of adaptive changes) host-virus arms races alter the proteins involved. Just as extreme amino acid conservation can serve to identify key immutable residues in enzymes, positively selected residues point to molecular recognition interfaces between host and viral proteins that have adapted and counter-adapted in a long series of classical Red Queen conflicts. Common rules for the strategies employed by both hosts and viruses have emerged from case studies of innate immunity genes in primates. We are now poised to use these rules to transition from a retrospective view of host-virus arms races to specific predictions about which host genes face pathogen antagonism and how those genetic conflicts transform host and virus evolution.

Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation.

Inflammasomes are cytosolic multi-protein complexes that initiate immune responses to infection by recruiting and activating the Caspase-1 protease. Human NLRP1 was the first protein shown to form an inflammasome, but its physiological mechanism of activation remains unknown. Recently, specific variants of mouse and rat NLRP1 were found to be activated upon N-terminal cleavage by the anthrax lethal factor protease. However, agonists for other NLRP1 variants, including human NLRP1, are not known, and it remains unclear if they are also activated by proteolysis. Here we demonstrate that two mouse NLRP1 paralogs (NLRP1AB6 and NLRP1BB6) are also activated by N-terminal proteolytic cleavage. We also demonstrate that proteolysis within a specific N-terminal linker region is sufficient to activate human NLRP1. Evolutionary analysis of primate NLRP1 shows the linker/cleavage region has evolved under positive selection, indicative of pathogen-induced selective pressure. Collectively, these results identify proteolysis as a general mechanism of NLRP1 inflammasome activation that appears to be contributing to the rapid evolution of NLRP1 in rodents and primates.

ICTV Virus Taxonomy Profile: Polyomaviridae.

The Polyomaviridae is a family of small, non-enveloped viruses with circular dsDNA genomes of approximately 5 kbp. The family includes four genera whose members have restricted host range, infecting mammals and birds. Polyomavirus genomes have also been detected recently in fish. Merkel cell polyomavirus and raccoon polyomavirus are associated with cancer in their host; other members are human and veterinary pathogens. Clinical manifestations are obvious in immunocompromised patients but not in healthy individuals. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Polyomaviridae, which is available at www.ictv.global/report/polyomaviridae.

Rapid evolution of PARP genes suggests a broad role for ADP-ribosylation in host-virus conflicts.

Post-translational protein modifications such as phosphorylation and ubiquitinylation are common molecular targets of conflict between viruses and their hosts. However, the role of other post-translational modifications, such as ADP-ribosylation, in host-virus interactions is less well characterized. ADP-ribosylation is carried out by proteins encoded by the PARP (also called ARTD) gene family. The majority of the 17 human PARP genes are poorly characterized. However, one PARP protein, PARP13/ZAP, has broad antiviral activity and has evolved under positive (diversifying) selection in primates. Such evolution is typical of domains that are locked in antagonistic 'arms races' with viral factors. To identify additional PARP genes that may be involved in host-virus interactions, we performed evolutionary analyses on all primate PARP genes to search for signatures of rapid evolution. Contrary to expectations that most PARP genes are involved in 'housekeeping' functions, we found that nearly one-third of PARP genes are evolving under strong recurrent positive selection. We identified a >300 amino acid disordered region of PARP4, a component of cytoplasmic vault structures, to be rapidly evolving in several mammalian lineages, suggesting this region serves as an important host-pathogen specificity interface. We also found positive selection of PARP9, 14 and 15, the only three human genes that contain both PARP domains and macrodomains. Macrodomains uniquely recognize, and in some cases can reverse, protein mono-ADP-ribosylation, and we observed strong signatures of recurrent positive selection throughout the macro-PARP macrodomains. Furthermore, PARP14 and PARP15 have undergone repeated rounds of gene birth and loss during vertebrate evolution, consistent with recurrent gene innovation. Together with previous studies that implicated several PARPs in immunity, as well as those that demonstrated a role for virally encoded macrodomains in host immune evasion, our evolutionary analyses suggest that addition, recognition and removal of ADP-ribosylation is a critical, underappreciated currency in host-virus conflicts.

A taxonomy update for the family Polyomaviridae.

Many distinct polyomaviruses infecting a variety of vertebrate hosts have recently been discovered, and their complete genome sequence could often be determined. To accommodate this fast-growing diversity, the International Committee on Taxonomy of Viruses (ICTV) Polyomaviridae Study Group designed a host- and sequence-based rationale for an updated taxonomy of the family Polyomaviridae. Applying this resulted in numerous recommendations of taxonomical revisions, which were accepted by the Executive Committee of the ICTV in December 2015. New criteria for definition and creation of polyomavirus species were established that were based on the observed distance between large T antigen coding sequences. Four genera (Alpha-, Beta, Gamma- and Deltapolyomavirus) were delineated that together include 73 species. Species naming was made as systematic as possible - most species names now consist of the binomial name of the host species followed by polyomavirus and a number reflecting the order of discovery. It is hoped that this important update of the family taxonomy will serve as a stable basis for future taxonomical developments.

A solution to limited genomic capacity: using adaptable binding surfaces to assemble the functional HIV Rev oligomer on RNA.

Many ribonucleoprotein (RNP) complexes assemble into large, organized structures in which protein subunits are positioned by interactions with RNA and other proteins. Here we demonstrate that HIV Rev, constrained in size by a limited viral genome, also forms an organized RNP by assembling a homo-oligomer on the Rev response element (RRE) RNA. Rev subunits bind cooperatively to discrete RNA sites using an oligomerization domain and an adaptable protein-RNA interface, forming a complex with 500-fold higher affinity than the tightest single interaction. High-affinity binding correlates strongly with RNA export activity. Rev utilizes different surfaces of its alpha-helical RNA-binding domain to recognize several low-affinity binding sites, including the well-characterized stem IIB site and an additional site in stem IA. We propose that adaptable RNA-binding surfaces allow the Rev oligomer to assemble economically into a discrete, stable RNP and provide a mechanistic role for Rev oligomerization during the HIV life cycle.

Identification of an overprinting gene in Merkel cell polyomavirus provides evolutionary insight into the birth of viral genes.

Many viruses use overprinting (alternate reading frame utilization) as a means to increase protein diversity in genomes severely constrained by size. However, the evolutionary steps that facilitate the de novo generation of a novel protein within an ancestral ORF have remained poorly characterized. Here, we describe the identification of an overprinting gene, expressed from an Alternate frame of the Large T Open reading frame (ALTO) in the early region of Merkel cell polyomavirus (MCPyV), the causative agent of most Merkel cell carcinomas. ALTO is expressed during, but not required for, replication of the MCPyV genome. Phylogenetic analysis reveals that ALTO is evolutionarily related to the middle T antigen of murine polyomavirus despite almost no sequence similarity. ALTO/MT arose de novo by overprinting of the second exon of T antigen in the common ancestor of a large clade of mammalian polyomaviruses. Taking advantage of the low evolutionary divergence and diverse sampling of polyomaviruses, we propose evolutionary transitions that likely gave birth to this protein. We suggest that two highly constrained regions of the large T antigen ORF provided a start codon and C-terminal hydrophobic motif necessary for cellular localization of ALTO. These two key features, together with stochastic erasure of intervening stop codons, resulted in a unique protein-coding capacity that has been preserved ever since its birth. Our study not only reveals a previously undefined protein encoded by several polyomaviruses including MCPyV, but also provides insight into de novo protein evolution.

Structure of the E3 ligase CRL2-ZYG11B with substrates reveals the molecular basis for N-degron recognition and ubiquitination.

ZYG11B is a substrate specificity factor for Cullin-RING ubiquitin ligase (CRL2) involved in many biological processes, including Gly/N-degron pathways. Yet how the binding of ZYG11B with CRL2 is coupled to substrate recognition and ubiquitination is unknown. We present the Cryo-EM structures of the CRL2-ZYG11B holoenzyme alone and in complex with a Gly/N-peptide from the inflammasome-forming pathogen sensor NLRP1. The structures indicate ZYG11B folds into a Leucine-Rich Repeat followed by two armadillo repeat domains that promote assembly with CRL2 and recognition of NLRP1 Gly/N-degron. ZYG11B promotes activation of the NLRP1 inflammasome through recognition and subsequent ubiquitination of the NLRP1 Gly/N-degron revealed by viral protease cleavage. Our structural and functional data indicate that blocking ZYG11B recognition of the NLRP1 Gly/N-degron inhibits NLRP1 inflammasome activation by a viral protease. Overall, we show how the CRL2-ZYG11B E3 ligase complex recognizes Gly/N-degron substrates, including those that are involved in viral protease-mediated activation of the NLRP1 inflammasome.

HIV Rev response element (RRE) directs assembly of the Rev homooligomer into discrete asymmetric complexes.

RNA is a crucial structural component of many ribonucleoprotein (RNP) complexes, including the ribosome, spliceosome, and signal recognition particle, but the role of RNA in guiding complex formation is only beginning to be explored. In the case of HIV, viral replication requires assembly of an RNP composed of the Rev protein homooligomer and the Rev response element (RRE) RNA to mediate nuclear export of unspliced viral mRNAs. Assembly of the functional Rev-RRE complex proceeds by cooperative oligomerization of Rev on the RRE scaffold and utilizes both protein-protein and protein-RNA interactions to organize complexes with high specificity. The structures of the Rev protein and a peptide-RNA complex are known, but the complete RNP is not, making it unclear to what extent RNA defines the composition and architecture of Rev-RNA complexes. Here we show that the RRE controls the oligomeric state and solubility of Rev and guides its assembly into discrete Rev-RNA complexes. SAXS and EM data were used to derive a structural model of a Rev dimer bound to an essential RRE hairpin and to visualize the complete Rev-RRE RNP, demonstrating that RRE binding drives assembly of Rev homooligomers into asymmetric particles, reminiscent of the role of RNA in organizing more complex RNP machines, such as the ribosome, composed of many different protein subunits. Thus, the RRE is not simply a passive scaffold onto which proteins bind but instead actively defines the protein composition and organization of the RNP.