A Sweep of Earth's Virome Reveals Host-Guided Viral Protein Structural Mimicry and Points to Determinants of Human Disease.

Viruses deploy genetically encoded strategies to coopt host machinery and support viral replicative cycles. Here, we use protein structure similarity to scan for molecular mimicry, manifested by structural similarity between viral and endogenous host proteins, across thousands of cataloged viruses and hosts spanning broad ecological niches and taxonomic range, including bacteria, plants and fungi, invertebrates, and vertebrates. This survey identified over 6,000,000 instances of structural mimicry; more than 70% of viral mimics cannot be discerned through protein sequence alone. We demonstrate that the manner and degree to which viruses exploit molecular mimicry varies by genome size and nucleic acid type and identify 158 human proteins that are mimicked by coronaviruses, providing clues about cellular processes driving pathogenesis. Our observations point to molecular mimicry as a pervasive strategy employed by viruses and indicate that the protein structure space used by a given virus is dictated by the host proteome. A record of this paper's transparent peer review process is included in the Supplemental Information.

Transcriptional response modules characterize IL-1ß and IL-6 activity in COVID-19.

Dysregulated IL-1ß and IL-6 responses have been implicated in the pathogenesis of severe Coronavirus Disease 2019 (COVID-19). Innovative approaches for evaluating the biological activity of these cytokines in vivo are urgently needed to complement clinical trials of therapeutic targeting of IL-1ß and IL-6 in COVID-19. We show that the expression of IL-1ß or IL-6 inducible transcriptional signatures (modules) reflects the bioactivity of these cytokines in immunopathology modelled by juvenile idiopathic arthritis (JIA) and rheumatoid arthritis. In COVID-19, elevated expression of IL-1ß and IL-6 response modules, but not the cytokine transcripts themselves, is a feature of infection in the nasopharynx and blood but is not associated with severity of COVID-19 disease, length of stay, or mortality. We propose that IL-1ß and IL-6 transcriptional response modules provide a dynamic readout of functional cytokine activity in vivo, aiding quantification of the biological effects of immunomodulatory therapies in COVID-19.

Transcriptional response modules characterise IL-1ß and IL-6 activity in COVID-19.

Dysregulated IL-1ß and IL-6 responses have been implicated in the pathogenesis of severe Coronavirus Disease 2019 (COVID-19). Innovative approaches for evaluating the biological activity of these cytokines in vivo are urgently needed to complement clinical trials of therapeutic targeting of IL-1ß and IL-6 in COVID-19. We show that the expression of IL-1ß or IL-6 inducible transcriptional signatures (modules) reflects the bioactivity of these cytokines in immunopathology modelled by juvenile idiopathic arthritis (JIA) and rheumatoid arthritis. In COVID-19, elevated expression of IL-1ß and IL-6 response modules, but not the cytokine transcripts themselves, is a feature of infection in the nasopharynx and blood, but is not associated with severity of COVID-19 disease, length of stay or mortality. We propose that IL-1ß and IL-6 transcriptional response modules provide a dynamic readout of functional cytokine activity in vivo, aiding quantification of the biological effects of immunomodulatory therapies in COVID-19.

Immune complement and coagulation dysfunction in adverse outcomes of SARS-CoV-2 infection.

Understanding the pathophysiology of SARS-CoV-2 infection is critical for therapeutic and public health strategies. Viral-host interactions can guide discovery of disease regulators, and protein structure function analysis points to several immune pathways, including complement and coagulation, as targets of coronaviruses. To determine whether conditions associated with dysregulated complement or coagulation systems impact disease, we performed a retrospective observational study and found that history of macular degeneration (a proxy for complement-activation disorders) and history of coagulation disorders (thrombocytopenia, thrombosis and hemorrhage) are risk factors for SARS-CoV-2-associated morbidity and mortality-effects that are independent of age, sex or history of smoking. Transcriptional profiling of nasopharyngeal swabs demonstrated that in addition to type-I interferon and interleukin-6-dependent inflammatory responses, infection results in robust engagement of the complement and coagulation pathways. Finally, in a candidate-driven genetic association study of severe SARS-CoV-2 disease, we identified putative complement and coagulation-associated loci including missense, eQTL and sQTL variants of critical complement and coagulation regulators. In addition to providing evidence that complement function modulates SARS-CoV-2 infection outcome, the data point to putative transcriptional genetic markers of susceptibility. The results highlight the value of using a multimodal analytical approach to reveal determinants and predictors of immunity, susceptibility and clinical outcome associated with infection.

Transcriptional response modules characterise IL-1 and IL-6 activity in COVID-19

Dysregulated IL-1{beta} and IL-6 responses have been implicated in the pathogenesis of severe Coronavirus Disease 2019 (COVID-19). Innovative approaches for evaluating the biological activity of these cytokines in vivo are urgently needed to complement clinical trials of therapeutic targeting of IL-1{beta} and IL-6 in COVID-19. We show that the expression of IL-1{beta} or IL-6 inducible transcriptional signatures (modules) reflects the bioactivity of these cytokines in immunopathology modelled by juvenile idiopathic arthritis (JIA) and rheumatoid arthritis. In COVID-19, elevated expression of IL-1{beta} and IL-6 response modules, but not the cytokine transcripts themselves, is a feature of infection in the nasopharynx and blood, but is not associated with severity of COVID-19 disease, length of stay or mortality. We propose that IL-1{beta} and IL-6 transcriptional response modules provide a dynamic readout of functional cytokine activity in vivo, aiding quantification of the biological effects of immunomodulatory therapies in COVID-19.

Identification of Immune complement function as a determinant of adverse SARS-CoV-2 infection outcome.

Understanding the pathophysiology of SARS-CoV-2 infection is critical for therapeutics and public health intervention strategies. Viral-host interactions can guide discovery of regulators of disease outcomes, and protein structure function analysis points to several immune pathways, including complement and coagulation, as targets of the coronavirus proteome. To determine if conditions associated with dysregulation of the complement or coagulation systems impact adverse clinical outcomes, we performed a retrospective observational study of 11,116 patients who presented with suspected SARS-CoV-2 infection. We found that history of macular degeneration (a proxy for complement activation disorders) and history of coagulation disorders (thrombocytopenia, thrombosis, and hemorrhage) are risk factors for morbidity and mortality in SARS-CoV-2 infected patients - effects that could not be explained by age, sex, or history of smoking. Further, transcriptional profiling of nasopharyngeal (NP) swabs from 650 control and SARS-CoV-2 infected patients demonstrated that in addition to innate Type-I interferon and IL-6 dependent inflammatory immune responses, infection results in robust engagement and activation of the complement and coagulation pathways. Finally, we conducted a candidate driven genetic association study of severe SARS-CoV-2 disease. Among the findings, our scan identified putative complement and coagulation associated loci including missense, eQTL and sQTL variants of critical regulators of the complement and coagulation cascades. In addition to providing evidence that complement function modulates SARS-CoV-2 infection outcome, the data point to putative transcriptional genetic markers of susceptibility. The results highlight the value of using a multi-modal analytical approach, combining molecular information from virus protein structure-function analysis with clinical informatics, transcriptomics, and genomics to reveal determinants and predictors of immunity, susceptibility, and clinical outcome associated with infection.

Identification of Immune complement function as a determinant of adverse SARS-CoV-2 infection outcome

SO_SCPLOWUMMARYC_SCPLOWUnderstanding the pathophysiology of SARS-CoV-2 infection is critical for therapeutics and public health intervention strategies. Viral-host interactions can guide discovery of regulators of disease outcomes, and protein structure function analysis points to several immune pathways, including complement and coagulation, as targets of the coronavirus proteome. To determine if conditions associated with dysregulation of the complement or coagulation systems impact adverse clinical outcomes, we performed a retrospective observational study of 11,116 patients who presented with suspected SARS-CoV-2 infection. We found that history of macular degeneration (a proxy for complement activation disorders) and history of coagulation disorders (thrombocytopenia, thrombosis, and hemorrhage) are risk factors for morbidity and mortality in SARS-CoV-2 infected patients - effects that could not be explained by age, sex, or history of smoking. Further, transcriptional profiling of nasopharyngeal (NP) swabs from 650 control and SARS-CoV-2 infected patients demonstrated that in addition to innate Type-I interferon and IL-6 dependent inflammatory immune responses, infection results in robust engagement and activation of the complement and coagulation pathways. Finally, we conducted a candidate driven genetic association study of severe SARS-CoV-2 disease. Among the findings, our scan identified putative complement and coagulation associated loci including missense, eQTL and sQTL variants of critical regulators of the complement and coagulation cascades. In addition to providing evidence that complement function modulates SARS-CoV-2 infection outcome, the data point to putative transcriptional genetic markers of susceptibility. The results highlight the value of using a multi-modal analytical approach, combining molecular information from virus protein structure-function analysis with clinical informatics, transcriptomics, and genomics to reveal determinants and predictors of immunity, susceptibility, and clinical outcome associated with infection.

A Structure-Informed Atlas of Human-Virus Interactions.

While knowledge of protein-protein interactions (PPIs) is critical for understanding virus-host relationships, limitations on the scalability of high-throughput methods have hampered their identification beyond a number of well-studied viruses. Here, we implement an in silico computational framework (pathogen host interactome prediction using structure similarity [P-HIPSTer]) that employs structural information to predict ∼282,000 pan viral-human PPIs with an experimental validation rate of ∼76%. In addition to rediscovering known biology, P-HIPSTer has yielded a series of new findings: the discovery of shared and unique machinery employed across human-infecting viruses, a likely role for ZIKV-ESR1 interactions in modulating viral replication, the identification of PPIs that discriminate between human papilloma viruses (HPVs) with high and low oncogenic potential, and a structure-enabled history of evolutionary selective pressure imposed on the human proteome. Further, P-HIPSTer enables discovery of previously unappreciated cellular circuits that act on human-infecting viruses and provides insight into experimentally intractable viruses.

Negative Regulation of Cytosolic Sensing of DNA.

In mammals, cytosolic detection of nucleic acids is critical in initiating innate antiviral responses against invading pathogens (like bacteria, viruses, fungi and parasites). These programs are mediated by multiple cytosolic and endosomal sensors and adaptor molecules (c-GAS/STING axis and TLR9/MyD88 axis, respectively) and lead to the production of type I interferons (IFNs), pro-inflammatory cytokines, and chemokines. While the identity and role of multiple pattern recognition receptors (PRRs) have been elucidated, such immune surveillance systems must be tightly regulated to limit collateral damage and prevent aberrant responses to self- and non-self-nucleic acids. In this review, we discuss recent advances in our understanding of how cytosolic sensing of DNA is controlled during inflammatory immune responses.

Germ-Cell-Specific Inflammasome Component NLRP14 Negatively Regulates Cytosolic Nucleic Acid Sensing to Promote Fertilization.

Cytosolic sensing of nucleic acids initiates tightly regulated programs to limit infection. Oocyte fertilization represents a scenario wherein inappropriate responses to exogenous yet non-pathogen-derived nucleic acids would have negative consequences. We hypothesized that germ cells express negative regulators of nucleic acid sensing (NAS) in steady state and applied an integrated data-mining and functional genomics approach to identify a rheostat of DNA and RNA sensing-the inflammasome component NLRP14. We demonstrated that NLRP14 interacted physically with the nucleic acid sensing pathway and targeted TBK1 (TANK binding kinase 1) for ubiquitination and degradation. We further mapped domains in NLRP14 and TBK1 that mediated the inhibitory function. Finally, we identified a human nonsense germline variant associated with male sterility that results in loss of NLRP14 function and hyper-responsiveness to nucleic acids. The discovery points to a mechanism of nucleic acid sensing regulation that may be of particular importance in fertilization.

A High-Throughput Strategy for Dissecting Mammalian Genetic Interactions.

Comprehensive delineation of complex cellular networks requires high-throughput interrogation of genetic interactions. To address this challenge, we describe the development of a multiplex combinatorial strategy to assess pairwise genetic interactions using CRISPR-Cas9 genome editing and next-generation sequencing. We characterize the performance of combinatorial genome editing and analysis using different promoter and gRNA designs and identified regions of the chimeric RNA that are compatible with next-generation sequencing preparation and quantification. This approach is an important step towards elucidating genetic networks relevant to human diseases and the development of more efficient Cas9-based therapeutics.

CHMP6 and VPS4A mediate the recycling of Ras to the plasma membrane to promote growth factor signaling.

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III (Endosomal Sorting Complex Required for Transport-III), were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding loop, and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound to N-Ras but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked epidermal growth factor receptor (EGFR) recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling.

Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells.

Cancer cells within individual tumors often exist in distinct phenotypic states that differ in functional attributes. While cancer cell populations typically display distinctive equilibria in the proportion of cells in various states, the mechanisms by which this occurs are poorly understood. Here, we study the dynamics of phenotypic proportions in human breast cancer cell lines. We show that subpopulations of cells purified for a given phenotypic state return towards equilibrium proportions over time. These observations can be explained by a Markov model in which cells transition stochastically between states. A prediction of this model is that, given certain conditions, any subpopulation of cells will return to equilibrium phenotypic proportions over time. A second prediction is that breast cancer stem-like cells arise de novo from non-stem-like cells. These findings contribute to our understanding of cancer heterogeneity and reveal how stochasticity in single-cell behaviors promotes phenotypic equilibrium in populations of cancer cells.

Systems biology approaches to dissect mammalian innate immunity.

Advances in experimental tools have allowed for the systematic identification of components and biological processes as well as quantification of their activities over time. Together with computational analysis, these measurement and perturbation technologies have given rise to the field of systems biology, which seeks to discover, analyze and model the interactions of physical components in a biological system. Although in its infancy, recent application of this approach has resulted in novel insights into the machinery that regulates and modifies innate immune cell functions. Here, we summarize contributions that have been made through the unbiased interrogation of the mammalian innate immune system, emphasizing the importance of integrating orthogonal datasets into models. To enable application of approaches more broadly, however, a concerted effort across the immunology community to develop reagent and tool platforms will be required.

A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection.

During the course of a viral infection, viral proteins interact with an array of host proteins and pathways. Here, we present a systematic strategy to elucidate the dynamic interactions between H1N1 influenza and its human host. A combination of yeast two-hybrid analysis and genome-wide expression profiling implicated hundreds of human factors in mediating viral-host interactions. These factors were then examined functionally through depletion analyses in primary lung cells. The resulting data point to potential roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of RNA-binding proteins, components of WNT signaling, and viral polymerase subunits. This multilayered approach provides a comprehensive and unbiased physical and regulatory model of influenza-host interactions and demonstrates a general strategy for uncovering complex host-pathogen relationships.

Spatial interactions in amblyopia: effects of stimulus parameters and amblyopia type.

Adults with amblyopia were recently shown to perform abnormally in tasks requiring integration of local features into global percepts. Moreover, spatial interactions in amblyopic patients, though often found to be abnormal, showed marked variability. Here we measured collinear lateral interactions using Gabor patches in a large number of amblyopic (N=75) and normal subjects (N=25), testing four spatial frequencies (1.5, 3, 6, 9 cpd). We used the lateral masking paradigm, in which the contrast-detection threshold is measured in the presence of high-contrast flankers at different distances from a central target. Whereas in normal subjects spatial interaction patterns were evident across all spatial frequencies, amblyopic subjects showed abnormal spatial interactions and increasing deficiencies with increasing spatial frequencies. These abnormalities depended on the axis of astigmatism (in meridional amblyopia) and were more pronounced in strabismic than in anisometropic amblyopia. Spatial interactions were independent on the contrast-detection thresholds. Thus, adults with amblyopia might perform as well as normal observers for some stimulus parameters and abnormally for others. Our results indicate a close relationship between abnormal visual input to the visual cortex during development and abnormal functionality of the collinear spatial interactions in adults with amblyopia.

Cell phenotype specific kinetics of expression of intratracheally injected manganese superoxide dismutase-plasmid/liposomes (MnSOD-PL) during lung radioprotective gene therapy.

Intratracheal (IT) injection of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) complexes prior to whole lung irradiation of C57BL/6J mice provides significant protection from acute and chronic irradiation damage. We determined the duration of increased MnSOD biochemical activity and differential expression of a hemagglutinin (HA) epitope-tagged MnSOD transgene. HA-MnSOD-PL was IT injected at doses of 0-1000 microg, and mice were killed 1,2,3 or 4 days later. Other groups of mice were irradiated to 20 Gy to the pulmonary cavity 24 h after injection and killed at the same time points as non-irradiated mice. Both non-irradiated and irradiated groups of mice showed increased MnSOD biochemical activity with plasmid dose that plateaued at 100 microg of MnSOD plasmid DNA. In control mice, MnSOD biochemical activity decreased at 2, 3 or 4 days after injection. In irradiated mice, MnSOD biochemical activity decreased at day 2 but increased on days 3 and 4. HA-MnSOD expression decreased in broncheoalveolar macrophages and alveolar type-II cells 3 days after injection in non-irradiated and irradiated mice, but remained elevated in endothelial and epithelial cells past 4 days. The data provide a rationale for every second-day administration of intrapulmonary MnSOD-PL in clinical trials of radioprotective gene therapy. This should be sufficient to provide radioprotection during radiation treatments.

A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If).

We describe a new congenital disorder of glycosylation, CDG-If. The patient has severe psychomotor retardation, seizures, failure to thrive, dry skin and scaling with erythroderma, and impaired vision. CDG-If is caused by a defect in the gene MPDU1, the human homologue of hamster Lec35, and is the first disorder to affect the use, rather than the biosynthesis, of donor substrates for lipid-linked oligosaccharides. This leads to the synthesis of incomplete and poorly transferred precursor oligosaccharides lacking both mannose and glucose residues. The patient has a homozygous point mutation (221T-->C, L74S) in a semiconserved amino acid of MPDU1. Chinese hamster ovary Lec35 cells lack a functional Lec35 gene and synthesize truncated lipid-linked oligosaccharides similar to the patient's. They lack glucose and mannose residues donated by Glc-P-Dol and Man-P-Dol. Transfection with the normal human MPDU1 allele nearly completely restores normal glycosylation, whereas transfection with the patient's MPDU1 allele only weakly restores normal glycosylation. This work provides a new clinical picture for another CDG that may involve synthesis of multiple types of glycoconjugates.

Disentangling signal from noise in visual contrast discrimination.

Human ability to detect stimulus changes (Delta C) decreases with increasing reference level (C). Because detection performance reflects the signal-to-noise ratio within the relevant sensory brain module, this behavior can be accounted for in two extreme ways: first, the internal response change Delta R evoked by a constant Delta C decreases with C (that is, the transducer R = f(C) displays a compressive nonlinearity), whereas the internal noise is independent of R; second, Delta R is constant with C but the noise level increases with R. A newly discovered constraint on human decision-making helps solve this century-old problem: in a detection task where multiple changes occur with equal probabilities, observers use a unique response criterion to decide whether a change has occurred. For contrast discrimination, our results supported the first account above: human performance was limited by the contrast transducer nonlinearity and an almost constant noise.