Cell

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit. Highlights{middle dot} ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species {middle dot} Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways {middle dot} Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function {middle dot} Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection

Understanding how nervous systems mediate responses to sensation requires whole-body maps of periphery-to-brain connections. Octopuses exemplify this challenge with distributed control of eight arms and hundreds of suckers, yet their long-range microanatomical wiring remains elusive due to limitations in microscopy. We extend histotomography (Ding et al. 2019), a form of soft tissue microCT customized for volumetric characterization of cells and tissues, to centimeter range with a custom micro-CT imaging system (Ding et al., 2019). With its 10-mm field of view and 0.7-{micro}m isotropic voxels we created a high-resolution digital intact small octopus. This multi-tissue 3D blueprint enabled us to (i) elucidate previously uncharacterized chemotactile pathways from the suckers to the brain, (ii) discern subdivisions of the nerve ring connecting neighboring arms, and (iii) segment over 300 structures across organ systems at histology-like resolution. We release the labeled interactive digital specimen to facilitate collaborative whole-organism phenotyping as a practical foundation for digital organismal biology. ONE-SENTENCE SUMMARYWhole-body 3D histology reveals neural and organ architecture throughout a small octopus.

We present Microbe-seq, a high-throughput single-microbe method that yields strain-resolved genomes from complex microbial communities. We encapsulate individual microbes into droplets with microfluidics and liberate their DNA, which we amplify, tag with droplet-specific barcodes, and sequence. We use Microbe-seq to explore the human gut microbiome; we collect stool samples from a single individual, sequence over 20,000 microbes, and reconstruct nearly-complete genomes of almost 100 bacterial species, including several with multiple subspecies strains. We use these genomes to probe genomic signatures of microbial interactions: we reconstruct the horizontal gene transfer (HGT) network within the individual and observe far greater exchange within the same bacterial phylum than between different phyla. We probe bacteria-virus interactions; unexpectedly, we identify a significant in vivo association between crAssphage, an abundant bacteriophage, and a single strain of Bacteroides vulgatus. Microbe-seq contributes high-throughput culture-free capabilities to investigate genomic blueprints of complex microbial communities with single-microbe resolution.

Prime-boost regimens for COVID-19 vaccines elicit poor antibody responses against Omicron-based variants and employ frequent boosters to maintain antibody levels. We present a natural infection-mimicking technology that combines features of mRNA- and protein nanoparticle-based vaccines through encoding self-assembling enveloped virus-like particles (eVLPs). eVLP assembly is achieved by inserting an ESCRT- and ALIX-binding region (EABR) into the SARS-CoV-2 spike cytoplasmic tail, which recruits ESCRT proteins to induce eVLP budding from cells. Purified spike-EABR eVLPs presented densely-arrayed spikes and elicited potent antibody responses in mice. Two immunizations with mRNA-LNP encoding spike-EABR elicited potent CD8+ T-cell responses and superior neutralizing antibody responses against original and variant SARS-CoV-2 compared to conventional spike-encoding mRNA-LNP and purified spike-EABR eVLPs, improving neutralizing titers >10-fold against Omicron-based variants for three months post-boost. Thus, EABR technology enhances potency and breadth of vaccine-induced responses through antigen presentation on cell surfaces and eVLPs, enabling longer-lasting protection against SARS-CoV-2 and other viruses.

The extracellular space is a critical environment for discriminating self versus non-self nucleic acids and initiating the appropriate immune responses through signaling cascades to relay information about extracellular nucleic acids. Here, we provide evidence that oxidized mitochondrial DNA is tethered to the surface of select mammalian cells through cell surface proteins and heparan sulfate proteoglycans. We demonstrate that cell surface DNA accumulates in large clusters that partially overlap with domains enriched in RNA binding proteins. Finally, we show that human and murine B cell surfaces contain DNA that can be cleared by the secreted nuclease DNASE1L3, and that patients with a DNASE1L3 missense variant associated with increased risk for autoimmune disease harbor increased levels of surface DNA on B and T cells. Taken together, this work expands the scope of cell surface nucleic acid biology and provides a mechanistic link between cell surface molecules and DNA targeting in autoimmune disease.

The skin colonist Staphylococcus epidermidis elicits a potent antibody response that can be redirected against an antigen of interest, but this process relies on genetic engineering.1 Here, by adapting bioorthogonal chemistry methods to conjugate antigens to the cell surface, we make the process of generating a commensal vaccine rapid and efficient. A wide variety of bacteria displaying tetanus toxin fragment C (TTFC) elicit an antibody response when applied to mice topically, indicating that the inductive process is not limited to colonists. Colonization occurs at two different sites, the skin and nostril; by colonizing each with S. epidermidis-TTFC, we show that skin colonization yields a moderate IgG response, while nostril colonization elicits a highly potent systemic IgG response and an exuberant IgA response in the nostrils, lungs, and intestine. Two lines of evidence are consistent with the nasal-associated lymphoid tissue (NALT) as the inductive site for nostril colonization: imaging suggesting robust bacterial translocation, and the induction of commensal-specific B cells following colonization. On the skin, TTFC must be conjugated to live S. epidermidis to elicit an antibody response; in the nostrils, live S. epidermidis-TTFC and S. epidermidis mixed with TTFC are equally potent. Commensal vaccination yields a robust response in pet shop mice, and chemical conjugation facilitates antibody responses to a broad array of antigens, including a whole viral capsid and a rotavirus immunogen. Collectively, these findings provide compelling evidence of a translational path for commensal vaccines.

We have created a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities across 3 years. This is the first systematic, worldwide study cataloging the urban microbial ecosystem. We identify taxonomically-defined microorganisms collected across three years. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics AMR markers, and novel genetic elements, including 10,928 viral, 1302 bacteria, and 2 archaea novel species. We identify 4,424 species of urban microorganisms and a consistent "core" of 31 species found in nearly all samples that is largely distinct from any human commensal microbiome. Profiles of AMR genes show geographic variation in type and density. Together, these results constitute a high-resolution, global metagenomic atlas, which enables the discovery of new genetic components, highlights potential forensic applications, and provides an essential first draft of the global AMR burden of the worlds cities.

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.

Many important biological functions arise not from single genes, but from complex interactions encoded by entire genomes. Genome language models have emerged as a promising strategy for designing biological systems, but their ability to generate functional sequences at the scale of whole genomes has remained untested. Here, we report the first generative design of viable bacteriophage genomes. We leveraged frontier genome language models, Evo 1 and Evo 2, to generate whole-genome sequences with realistic genetic architectures and desirable host tropism, using the lytic phage {Phi}X174 as our design template. Experimental testing of AI-generated genomes yielded 16 viable phages with substantial evolutionary novelty. Cryo-electron microscopy revealed that one of the generated phages utilizes an evolutionarily distant DNA packaging protein within its capsid. Multiple phages demonstrate higher fitness than {Phi}X174 in growth competitions and in their lysis kinetics. A cocktail of the generated phages rapidly overcomes {Phi}X174-resistance in three E. coli strains, demonstrating the potential utility of our approach for designing phage therapies against rapidly evolving bacterial pathogens. This work provides a blueprint for the design of diverse synthetic bacteriophages and, more broadly, lays a foundation for the generative design of useful living systems at the genome scale.

Mitochondrial DNA (mtDNA) is the oldest and most prevalent source of human molecular phylogenetic reconstruction. Everyone alive today traces their matrilines to one female African ancestor who lived some 145 thousand years ago. For decades, PhyloTree provided researchers with a moderately sized reference tree (v17: n=24,275 sequences, n=5,438 branches). However, hundreds of thousands of sequences are available today, and since PhyloTrees retirement in 2016, there is a need for an actively maintained phylogenetic reference system. In addition, no currently published method can wield such a large de novo reconstruction while dealing with the rampant homoplasy seen in mtDNA and adhering to the accuracy standards expected in this well-studied field. In this paper we introduce Mitotree, the largest human phylogeny ever described (n{approx}330,000 complete sequences, n{approx}54,000 branches), and a new recursive phylogenetic pipeline for estimating it. We incorporate ancient DNA, relaxed clock age estimates (TMRCAs), and public databases (e.g., GenBank, 1000 Genomes, HGDP, and SGDP). We report approximately 180 previously unknown branches older than 30,000 years. The median TMRCA of terminal haplogroups is 2,000 years more recent than PhyloTree. Our pipeline offers a novel divide-and-conquer approach that tackles huge heuristic searches within tractable runtimes, provides high accuracy, and reasonable confidence estimates. Our validation found a false negative rate of 3.5%, and a false positive rate of 1.0%. Among the most striking findings are an [~]83 kya split of African L2e (the oldest in our dataset), the resolution of Otzis K1f into a clade with living descendants, new ethnic founder haplogroups (e.g., Jewish diaspora), and placement of historical figures such as Abraham Lincoln. To our knowledge, Mitotree is the largest de novo haplotype phylogeny built for humanity, and is a continually improved resource for academic, genealogical, and medical research.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

COVID-19 has had high incidence at institutions of higher education (IHE) in the United States, but the transmission dynamics in these settings are not well understood. It remains unclear to what extent IHE-associated outbreaks have contributed to transmission in nearby communities. We implemented high-density prospective genomic surveillance to investigate these dynamics at the University of Michigan-Ann Arbor and the surrounding community during the Fall 2020 semester (August 16th -November 24th). We sequenced complete SARS-CoV-2 genomes from 1659 individuals, including 468 students, representing 20% of cases in students and 25% of total confirmed cases in Washtenaw County over the study interval. Phylogenetic analysis identified over 200 introductions into the student population, most of which were not related to other student cases. There were two prolonged transmission clusters among students that spanned across multiple on-campus residences. However, there were very few genetic descendants of student clusters among non-students during a subsequent November wave of infections in the community. We conclude that outbreaks at the University of Michigan did not significantly contribute to the rise in Washtenaw County COVID-19 incidence during November 2020. These results provide valuable insights into the distinct transmission dynamics of SARS-CoV-2 among IHE populations and surrounding communities.

Tens of thousands of genetic variants shape human phenotypes, mostly by unknown cellular mechanisms. Here we describe Census-seq, a way to measure cellular phenotypes in cells from many people simultaneously. Analogous to pooled CRISPR screens but for natural variation, Census-seq associates cellular phenotypes to donors genotypes by quantifying the presence of each donors DNA in cell "villages" before and after sorting or selection for cellular traits of interest. Census-seq enables population-scale cell-biological phenotyping with low cost and high internal control. We demonstrate Census-seq through investigation of genetic effects on the SMN protein whose deficiency underlies spinal muscular atrophy (SMA). Census-seq quantified and mapped effects of many common alleles on SMN protein levels and response to SMN-targeted therapeutics, including a common, cryptic non-responder allele. We provide tools enabling population-scale cell experiments and explain how Census-seq can be used to map genetic effects on diverse cell phenotypes. O_FIG O_LINKSMALLFIG WIDTH=185 HEIGHT=200 SRC="FIGDIR/small/174383v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@169bf4borg.highwire.dtl.DTLVardef@18dd9c0org.highwire.dtl.DTLVardef@1d30ca5org.highwire.dtl.DTLVardef@e35bf0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICensus-seq reveals how inherited genetic variation affects cell phenotypes C_LIO_LIGenetic analysis of cellular traits in cell villages of >100 donors C_LIO_LICharacterizing human alleles that shape SMN protein expression and drug responses C_LIO_LIDevelopment of protocols and software to enable cellular population genetics C_LI

The extent of the devastation of the Black Death pandemic (1346-53) on European populations is known from documentary sources and its bacterial source illuminated by studies of ancient pathogen DNA. What has remained less understood is the effect of the pandemic on human mobility and genetic diversity at local scale in the context of the social stratification of medieval communities. Here we study 275 newly reported ancient genomes from later medieval and post-medieval Cambridgeshire, from individuals buried before, during, and after the Black Death. The majority of individuals examined had local genetic ancestries. Consistent with the function of the institutions, we found a lack of close relatives among the friars and the inmates of the hospital in contrast to their abundance in general urban and rural parish communities. Accounting for the genetic component for height accentuates the disparities between social groups in stature estimated from long bones, as a proxy for health and the quality of life. While we detect long-term shifts in local genetic ancestry in Cambridgeshire that either pre- or postdate the Black Death, we find no evidence of major changes in genetic ancestry nor, in contrast to recent claims, higher differentiation of immune loci between cohorts living before and after the Black Death.

The development of high-throughput single-cell RNA-sequencing (scRNA-Seq) methodologies has empowered the characterization of complex biological samples by dramatically increasing the number of constituent cells that can be examined concurrently. Nevertheless, these approaches typically recover substantially less information per-cell as compared to lower-throughput microtiter plate-based strategies. To uncover critical phenotypic differences among cells and effectively link scRNA-Seq observations to legacy datasets, reliable detection of phenotype-defining transcripts - such as transcription factors, affinity receptors, and signaling molecules - by these methods is essential. Here, we describe a substantially improved massively-parallel scRNA-Seq protocol we term Seq-Well S^3 (\"Second-Strand Synthesis\") that increases the efficiency of transcript capture and gene detection by up to 10- and 5-fold, respectively, relative to previous iterations, surpassing best-in-class commercial analogs. We first characterized the performance of Seq-Well S^3 in cell lines and PBMCs, and then examined five different inflammatory skin diseases, illustrative of distinct types of inflammation, to explore the breadth of potential immune and parenchymal cell states. Our work presents an essential methodological advance as well as a valuable resource for studying the cellular and molecular features that inform human skin inflammation.

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbors through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types where mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

The gut microbiome is a key modulator of immune and metabolic health. Human microbiome data is biased towards industrialized populations, providing limited understanding of the distinct and diverse non-industrialized microbiomes. Here, we performed ultra-deep metagenomic sequencing and strain cultivation on 351 fecal samples from the Hadza, hunter-gatherers in Tanzania, and comparative populations in Nepal and California. We recover 94,971 total genomes of bacteria, archaea, bacteriophages, and eukaryotes, 43% of which are absent from existing unified datasets. Analysis of in situ growth rates, genetic pN/pS signatures, high-resolution strain tracking, and 124 gut-resident species vanishing in industrialized populations reveals differentiating dynamics of the Hadza gut microbiome. Industrialized gut microbes are enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. This unparalleled view of the Hadza gut microbiome provides a valuable resource that expands our understanding of microbes capable of colonizing the human gut and clarifies the extensive perturbation brought on by the industrialized lifestyle.

Cells communicate with each other via receptor-ligand interactions on the cell surface. Here we describe a technology for lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to decode receptor specificity. Engineered lentiviral particles displaying specific ligands deliver fluorescent proteins into target cells upon cognate receptor-ligand interaction, without genome integration or transgene transcription. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. We develop an effective presentation strategy to capture interactions between B cell receptor (BCR) and intracellular antigen epitopes. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of TCR-antigen specificities, clonality, cell type, and cell states of individual T cells. ENTER-seq of patient blood samples after CMV infection reveals the viral epitopes that drive human effector memory T cell differentiation and inter-clonal phenotypic diversity that targets the same epitope. ENTER enables systematic discovery of receptor specificity, linkage to cell fates, and cell-specific delivery of gene or protein payloads. HIGHLIGHTSO_LIENTER displays ligands, deliver cargos, and records receptor specificity. C_LIO_LIENTER deorphanizes antigen recognition of TCR and BCR. C_LIO_LIENTER-seq maps TCR specificity, clonality and cell state in single cells. C_LIO_LIENTER-seq of patient sample decodes antiviral T cell memory. C_LI

The clinical state of diseased tissue is caused by complex intercellular processes that go beyond pairwise cell-cell interactions and are difficult to infer due to the combinatorial explosion of such high-dimensionality. We present context-dependent identification of spatial motifs (CISM), a two-step method to identify local cell structures associated with a disease state in single cell spatial data. First, for each tissue, CISM enumerates structures of enriched reoccurring multicellular patterns that define modular motifs in the multicellular network. Second, discriminative motifs are selected according to the context - their presence in patients at different clinical disease states. By applying CISM, we show that modular structures composed of as little as 3-5 cells and their relative spatial arrangement can encode differences in clinical disease states in cohorts of triple-negative breast cancer (TNBC) and melanoma patients. Machine learning validation indicated that discriminative motifs outperform state-of-the-art methods for disease state prediction while enabling interpretation of which interactions in what spatial context are associated with these predictions. CISM-derived discriminative motifs may define an intermediate spatial scale of abstraction and modularity in multicellular organization and function with broad applicability in the domain of spatial single cell omics and beyond.

Transposable elements are abundant in host genomes but are generally considered to be confined to the cell in which they are expressed, with the notable exception of endogenous retroviruses. Here, we identify a group of LTR retrotransposons that infect the germline from somatic cells within the Drosophila ovary, despite lacking the fusogenic Envelope protein typically required for retroviral entry. Instead, these elements encode a short transmembrane protein, sORF2, with structural features reminiscent of viral cell-cell fusogens. Through genetics, imaging, and electron microscopy, we show that sORF2 localizes to invasive somatic protrusions, enabling the direct transfer of retrotransposon capsids into the oocyte. Remarkably, sORF2-like proteins are widespread among insect retrotransposons and also occur in piscine nackednaviruses and avian picornaviruses. These findings reveal a noncanonical, Envelope-independent transmission mechanism shared by retrotransposons and non-enveloped viruses, offering important insights into host-pathogen evolution and soma-germline interactions.