Evolutionary and functional analyses reveal a role for the RHIM in tuning RIPK3 activity across vertebrates.

Receptor interacting protein kinases (RIPK) RIPK1 and RIPK3 play important roles in diverse innate immune pathways. Despite this, some RIPK1/3-associated proteins are absent in specific vertebrate lineages, suggesting that some RIPK1/3 functions are conserved while others are more evolutionarily labile. Here, we perform comparative evolutionary analyses of RIPK1-5 and associated proteins in vertebrates to identify lineage-specific rapid evolution of RIPK3 and RIPK1 and recurrent loss of RIPK3-associated proteins. Despite this, diverse vertebrate RIPK3 proteins are able to activate NF-κB and cell death in human cells. Additional analyses revealed a striking conservation of the RIP homotypic interaction motif (RHIM) in RIPK3, as well as other human RHIM-containing proteins. Interestingly, diversity in the RIPK3 RHIM can tune activation of NF-κB while retaining the ability to activate cell death. Altogether, these data suggest that NF-κB activation is a core, conserved function of RIPK3, and the RHIM can tailor RIPK3 function to specific needs within and between species.

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.

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.

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.

Structure of LRRK1 and mechanisms of autoinhibition and activation.

Leucine Rich Repeat Kinase 1 and 2 (LRRK1 and LRRK2) are homologs in the ROCO family of proteins in humans. Despite their shared domain architecture and involvement in intracellular trafficking, their disease associations are strikingly different: LRRK2 is involved in familial Parkinson's disease while LRRK1 is linked to bone diseases. Furthermore, Parkinson's disease-linked mutations in LRRK2 are typically autosomal dominant gain-of-function while those in LRRK1 are autosomal recessive loss-of-function. Here, to understand these differences, we solved cryo-EM structures of LRRK1 in its monomeric and dimeric forms. Both differ from the corresponding LRRK2 structures. Unlike LRRK2, which is sterically autoinhibited as a monomer, LRRK1 is sterically autoinhibited in a dimer-dependent manner. LRRK1 has an additional level of autoinhibition that prevents activation of the kinase and is absent in LRRK2. Finally, we place the structural signatures of LRRK1 and LRRK2 in the context of the evolution of the LRRK family of proteins.

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

Natural selection drives 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 acquisition of functions in immunity, metabolism, and reproduction via interdomain HGT (iHGT) from bacteria. We report that the nematode gene rml-3, which was acquired by iHGT from bacteria, enables exoskeleton resilience and protection against environmental toxins in C. elegans. Phylogenetic analysis reveals that diverse nematode RML-3 proteins form a single monophyletic clade most highly similar to bacterial enzymes that biosynthesize L-rhamnose to build cell wall polysaccharides. C. elegans rml-3 is regulated in developing seam cells by heat stress and stress-resistant dauer stage. Importantly, rml-3 deficiency impairs cuticle integrity, barrier functions and organismal stress resilience, phenotypes that are rescued by exogenous L-rhamnose. We propose that iHGT of an ancient bacterial rml-3 homolog enables L-rhamnose biosynthesis in nematodes that facilitates cuticle integrity and organismal resilience in adaptation to environmental stresses during evolution. These findings highlight the remarkable contribution of iHGT on metazoan evolution that is conferred by the domestication of bacterial genes.

Tripping the wire: sensing of viral protease activity by CARD8 and NLRP1 inflammasomes.

Host innate immune sensors are vital for the initial detection of pathogen infection. Such sensors thus need to constantly adapt in escalating evolutionary arms races with pathogens. Recently, two inflammasome-forming proteins, CARD8 and NLRP1, have emerged as innate immune sensors for the enzymatic activity of virus-encoded proteases. When cleaved within a rapidly evolving 'tripwire' region, CARD8 and NLRP1 assemble into inflammasomes that initiate pyroptotic cell death and pro-inflammatory cytokine release as a form of effector-triggered immunity. Short motifs in the CARD8 and NLRP1 tripwires mimic the protease-specific cleavage sites of picornaviruses, coronaviruses, and HIV-1, providing virus-specific sensing that can rapidly change between closely related hosts and within the human population. Recent work highlights the evolutionary arms races between viral proteases and NLRP1 and CARD8, including insights into the mechanisms of inflammasome activation, host diversity of viral sensing, and means that viruses have evolved to avoid tripping the wire.

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.

Recurrent Loss of Macrodomain Activity in Host Immunity and Viral Proteins.

Protein post-translational modifications (PTMs) are an important battleground in the evolutionary arms races that are waged between the host innate immune system and viruses. One such PTM, ADP-ribosylation, has recently emerged as an important mediator of host antiviral immunity. Important for the host-virus conflict over this PTM is the addition of ADP-ribose by PARP proteins and removal of ADP-ribose by macrodomain-containing proteins. Interestingly, several host proteins, known as macroPARPs, contain macrodomains as well as a PARP domain, and these proteins are both important for the host antiviral immune response and evolving under very strong positive (diversifying) evolutionary selection. In addition, several viruses, including alphaviruses and coronaviruses, encode one or more macrodomains. Despite the presence of the conserved macrodomain fold, the enzymatic activity of many of these proteins has not been characterized. Here, we perform evolutionary and functional analyses to characterize the activity of macroPARP and viral macrodomains. We trace the evolutionary history of macroPARPs in metazoans and show that PARP9 and PARP14 contain a single active macrodomain, whereas PARP15 contains none. Interestingly, we also reveal several independent losses of macrodomain enzymatic activity within mammalian PARP14, including in the bat, ungulate, and carnivore lineages. Similar to macroPARPs, coronaviruses contain up to three macrodomains, with only the first displaying catalytic activity. Intriguingly, we also reveal the recurrent loss of macrodomain activity within the alphavirus group of viruses, including enzymatic loss in insect-specific alphaviruses as well as independent enzymatic losses in two human-infecting viruses. Together, our evolutionary and functional data reveal an unexpected turnover in macrodomain activity in both host antiviral proteins and viral proteins.

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.

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.

Co-opted genes of algal origin protect C. elegans against cyanogenic toxins.

Amygdalin is a cyanogenic glycoside enriched in the tissues of many edible plants, including seeds of stone fruits such as cherry (Prunus avium), peach (Prunus persica), and apple (Malus domestica). These plants biosynthesize amygdalin in defense against herbivore animals, as amygdalin generates poisonous cyanide upon plant tissue destruction.1,2,3,4 Poisonous to many animals, amygdalin-derived cyanide is detoxified by potent enzymes commonly found in bacteria and plants but not most animals.5 Here we show that the nematode C. elegans can detoxify amygdalin by a genetic pathway comprising cysl-1, egl-9, hif-1, and cysl-2. A screen of a natural product library for hypoxia-independent regulators of HIF-1 identifies amygdalin as a potent activator of cysl-2, a HIF-1 transcriptional target that encodes a cyanide detoxification enzyme in C. elegans. As a cysl-2 paralog similarly essential for amygdalin resistance, cysl-1 encodes a protein homologous to cysteine biosynthetic enzymes in bacteria and plants but functionally co-opted in C. elegans. We identify exclusively HIF-activating egl-9 mutations in a cysl-1 suppressor screen and show that cysl-1 confers amygdalin resistance by regulating HIF-1-dependent cysl-2 transcription to protect against amygdalin toxicity. Phylogenetic analysis indicates that cysl-1 and cysl-2 were likely acquired from green algae through horizontal gene transfer (HGT) and functionally co-opted in protection against amygdalin. Since acquisition, these two genes evolved division of labor in a cellular circuit to detect and detoxify cyanide. Thus, algae-to-nematode HGT and subsequent gene function co-option events may facilitate host survival and adaptation to adverse environmental stresses and biogenic toxins.

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 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, a methyltransferase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a possible target for future antiviral therapies. Importance: Similar to other coronaviruses, disruption of 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.

Antiviral function and viral antagonism of the rapidly evolving dynein activating adaptor NINL.

Viruses interact with the intracellular transport machinery to promote viral replication. Such host-virus interactions can drive host gene adaptation, leaving signatures of pathogen-driven evolution in host genomes. Here, we leverage these genetic signatures to identify the dynein activating adaptor, ninein-like (NINL), as a critical component in the antiviral innate immune response and as a target of viral antagonism. Unique among genes encoding components of active dynein complexes, NINL has evolved under recurrent positive (diversifying) selection, particularly in its carboxy-terminal cargo-binding region. Consistent with a role for NINL in host immunity, we demonstrate that NINL knockout cells exhibit an impaired response to interferon, resulting in increased permissiveness to viral replication. Moreover, we show that proteases encoded by diverse picornaviruses and coronaviruses cleave and disrupt NINL function in a host- and virus-specific manner. Our work reveals the importance of NINL in the antiviral response and the utility of using signatures of host-virus genetic conflicts to uncover new components of antiviral immunity and targets of viral antagonism.

Humans and viruses are locked in an evolutionary arms race. Viruses hijack cells, using their resources and proteins to build more viral particles; the cells fight back, calling in the immune system to fend off the attack. Both actors must constantly and quickly evolve to keep up with each other. This genetic conflict has been happening for millions of years, and the indelible marks it has left on genes can serve to uncover exactly how viruses interact with the organisms they invade. One hotspot in this host-virus conflict is the complex network of molecules that help to move cargo inside a cell. This system transports elements of the immune system, but viruses can also harness it to make more of themselves. Scientists still know very little about how viruses and the intracellular transport machinery interact, and how this impacts viral replication and the immune response. Stevens et al. therefore set out to identify new interactions between viruses and the transport system by using clues left in host genomes by evolution. They focused on dynein, a core component of this machinery which helps to haul molecular actors across a cell. To do so, dynein relies on adaptor molecules such as 'Ninein-like', or NINL for short. Closely examining the gene sequence for NINL across primates highlighted an evolutionary signature characteristic of host-virus genetic conflicts; this suggests that the protein may be used by viruses to reproduce, or by cells to fend off infection. And indeed, human cells lacking the NINL gene were less able to defend themselves, allowing viruses to grow much faster than normal. Further work showed that NINL was important for a major type of antiviral immune response. As a potential means to sabotage this defence mechanism, some viruses cleave NINL at specific sites and disrupt its role in intracellular transport. Better antiviral treatments are needed to help humanity resist old foes and new threats alike. The work by Stevens et al. demonstrates how the information contained in host genomes can be leveraged to understand what drives susceptibility to an infection, and to pinpoint molecular actors which could become therapeutic targets.

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 (3CL pro ) encoded by diverse coronaviruses, including 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 3CL pro , including 3CL pro -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 3CL pros , and instead enables sensing of 3C proteases (3C pro ) 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 intra-species variation in inflammasome-mediated viral sensing and immunopathology.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Running With Scissors: Evolutionary Conflicts Between Viral Proteases and the Host Immune System.

Many pathogens encode proteases that serve to antagonize the host immune system. In particular, viruses with a positive-sense single-stranded RNA genome [(+)ssRNA], including picornaviruses, flaviviruses, and coronaviruses, encode proteases that are not only required for processing viral polyproteins into functional units but also manipulate crucial host cellular processes through their proteolytic activity. Because these proteases must cleave numerous polyprotein sites as well as diverse host targets, evolution of these viral proteases is expected to be highly constrained. However, despite this strong evolutionary constraint, mounting evidence suggests that viral proteases such as picornavirus 3C, flavivirus NS3, and coronavirus 3CL, are engaged in molecular 'arms races' with their targeted host factors, resulting in host- and virus-specific determinants of protease cleavage. In cases where protease-mediated cleavage results in host immune inactivation, recurrent host gene evolution can result in avoidance of cleavage by viral proteases. In other cases, such as recently described examples in NLRP1 and CARD8, hosts have evolved 'tripwire' sequences that mimic protease cleavage sites and activate an immune response upon cleavage. In both cases, host evolution may be responsible for driving viral protease evolution, helping explain why viral proteases and polyprotein sites are divergent among related viruses despite such strong evolutionary constraint. Importantly, these evolutionary conflicts result in diverse protease-host interactions even within closely related host and viral species, thereby contributing to host range, zoonotic potential, and pathogenicity of viral infection. Such examples highlight the importance of examining viral protease-host interactions through an evolutionary lens.