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.

Development of a sterile insect technique as a control strategy for the Asian citrus psyllid: establishing the effect of sterilizing X-rays on fecundity, fertility, and survival.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a major pest of citrus due to its role as the vector of the bacterium that causes huanglongbing. In commercial citrus, ACP control currently relies on the application of insecticides, which may not be sustainable long-term, nor practical in urban areas. The sterile insect technique (SIT) is an alternative strategy in which large numbers of pests are reared, sterilized using radiation, and then released into the field to compete with wild individuals for matings, suppressing population growth. As a fundamental step toward the development of SIT for ACP, this study sought to identify the optimum radiation dose required to sterilize ACP without affecting their survival and mating capacity. Virgin adult ACP of both sexes were subjected to doses of X-ray irradiation ranging from 40 to 480 Gy, then paired with a nonirradiated mate and allowed to produce offspring. Fecundity was estimated as the number of eggs laid, and fertility as the proportion of those eggs that hatched. Females were more radio-sensitive than males, exhibiting a major drop in fecundity at even the lowest dose and 100% sterility at 80 Gy. In contrast, a fivefold higher dose (400 Gy) did not achieve complete sterility in males, with around 5% offspring survival. However, F1 progeny of males exposed to 320 Gy or higher were subsequently found to be 100% sterile. This confirmation of inherited sterility suggests that balancing the sterilizing effects of radiation against its mortality-inducing effects may warrant further evaluation.

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.

Test-retest reliability of quantitative sensory testing, active joint position sense, and functional hop testing in amateur adult athletes with unilateral anterior cruciate ligament reconstruction.

OBJECTIVES: The somatosensory system fulfils a critical role in functional knee joint stability (FKJS) by providing afferent feedback necessary for neuromuscular control. Individuals with anterior cruciate ligament reconstruction (ACLr) have altered somatosensory function. Somatosensory characteristics are assessed by proprioception and quantitative sensory testing. The purpose of the study was to examine intra-rater and inter-rater reliability of methods used to assess somatosensory characteristics and FKJS in amateur adult athletes with unilateral ACLr. DESIGN: Repeated measures. SETTING: University. PARTICIPANTS: 8 female, 4 male with unilateral autogenous ACLr. MAIN OUTCOME MEASURES: Bilateral measurements at 5 lower extremity locations and the anterior forearm: light touch (LT), vibration sense (VS), pressure pain threshold (PPT); knee active joint position sense (AJPS); adapted crossover hop for distance (ACHD). Intraclass correlation coefficients (ICC) determined reliability, defined as: poor (<0.50), moderate (0.50-0.75), good (0.75-0.90). RESULTS: ACLr-side intra-rater/inter-rater ICCs ranged: LT, -0.27-0.80/-0.01-0.84; VS, 0.12-0.90/0.25-0.90; PPT, 0.49-0.98/0.86-0.99; AJPS, 0.15-0.79/0.55-0.87; ACHD, 0.98/0.99. Uninjured-side intra-rater/inter-rater ICCs ranged: LT, 0.12-0.66/-0.09-0.64; VS, 0.35-0.89/0.05-0.81; PPT, 0.65-0.99/0.45-0.95; AJPS, 0.07-0.81/0.37-0.99; ACHD, 0.99/0.98. CONCLUSIONS: Intra-rater and inter-rater reliability was poor to good for both limbs. Overall, PPT and the ACHD demonstrated the highest ICCs. Some somatosensory assessments can be employed with confidence, while others should be used with caution.

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.

Progression of Xylella fastidiosa Infection in Grapevines Under Field Conditions.

The pathogen Xylella fastidiosa subsp. fastidiosa has circulated through California's vineyards since its introduction from Central America in the 1800s. This pathogen is responsible for a bacterial disease called Pierce's disease (PD) of grapevine. With no known cure, PD has had devastating effects on some vineyards. Important factors that impact disease severity and persistence include: the presence of insect vectors, grapevine cultivar, management, ecology, and winter temperatures. Removal of infected vines is critical for reducing pathogen spread but relies on accurate and rapid pathogen detection. In this study, we foster a greater understanding of disease symptom emergence by way of a 3-year field inoculation project in Napa Valley. Although PD emergence and symptom progression have been studied in greenhouse and experimental plots, there is a large knowledge gap in quantifying disease progression under commercial conditions. After inoculating 80 mature Vitis vinifera vines in April 2017, we measured bacterial populations and six symptom types at four locations within each plant throughout the subsequent three growing seasons. The main foci of the project were understanding X. fastidiosa movement through the plants, infection, overwinter curing, and symptom development. We observed greater winter recovery than expected, and shriveled grape clusters proved to be a more reliable early indication of infection than other more commonly used symptoms. Although there were differences among wine grape cultivars, this work suggests that disease progression in the field may not fit the paradigm of predominant leaf scorch and low recovery rates as neatly as has been previously believed.

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.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.