Human paraneoplastic antigen Ma2 (PNMA2) forms icosahedral capsids that can be engineered for mRNA delivery.

A number of endogenous genes in the human genome encode retroviral gag-like proteins, which were domesticated from ancient retroelements. The paraneoplastic Ma antigen (PNMA) family members encode a gag-like capsid domain, but their ability to assemble as capsids and traffic between cells remains mostly uncharacterized. Here, we systematically investigate human PNMA proteins and find that a number of PNMAs are secreted by human cells. We determine that PNMA2 forms icosahedral capsids efficiently but does not naturally encapsidate nucleic acids. We resolve the cryoelectron microscopy (cryo-EM) structure of PNMA2 and leverage the structure to design engineered PNMA2 (ePNMA2) particles with RNA packaging abilities. Recombinantly purified ePNMA2 proteins package mRNA molecules into icosahedral capsids and can function as delivery vehicles in mammalian cell lines, demonstrating the potential for engineered endogenous capsids as a nucleic acid therapy delivery modality.

Cell type-specific delivery by modular envelope design.

The delivery of genetic cargo remains one of the largest obstacles to the successful translation of experimental therapies, in large part due to the absence of targetable delivery vectors. Enveloped delivery modalities use viral envelope proteins, which determine tropism and induce membrane fusion. Here we develop DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design), a modular platform that consists of separate fusion and targeting components. To achieve high modularity and programmable cell type specificity, we develop multiple strategies to recruit or immobilize antibodies on the viral envelope, including a chimeric antibody binding protein and a SNAP-tag enabling the use of antibodies or other proteins as targeting molecules. Moreover, we show that fusogens from multiple viral families are compatible with DIRECTED and that DIRECTED components can target multiple delivery chassis (e.g., lentivirus and MMLV gag) to specific cell types, including primary human T cells in PBMCs and whole blood.

The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity.

TnpB is a member of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family, is harbored in transposons, and likely functions to maintain the transposon in genomes. Previously, it was shown that TnpB cleaves double- and single-stranded DNA substrates in an RNA-guided manner, but the biogenesis of the TnpB ribonucleoprotein (RNP) complex is unknown. Using in vitro purified apo TnpB, we demonstrate the ability of TnpB to generate guide omegaRNA (ωRNA) from its own mRNA through 5' processing. We also uncover a potential cis-regulatory mechanism whereby a region of the TnpB mRNA inhibits DNA cleavage by the RNP complex. We further expand the characterization of TnpB by examining ωRNA processing and RNA-guided nuclease activity in 59 orthologs spanning the natural diversity of the TnpB family. This work reveals a new functionality, ωRNA biogenesis, of TnpB, and characterizes additional members of this biotechnologically useful family of programmable enzymes.

Modularity and diversity of target selectors in Tn7 transposons.

To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, which we show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread.

Fanzor is a eukaryotic programmable RNA-guided endonuclease.

RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR-Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage1. Although a few eukaryotic RNA-guided systems have been studied, including RNA interference2 and ribosomal RNA modification3, it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported4,5. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity4,6. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins4,7, raising the possibility that eukaryotes are also equipped with CRISPR-Cas or OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolve the structure of Spizellomyces punctatus Fz at 2.7 Å using cryogenic electron microscopy, showing the conservation of core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life.

Exaptation of Inactivated Host Enzymes for Structural Roles in Orthopoxviruses and Novel Folds of Virus Proteins Revealed by Protein Structure Modeling.

Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds. IMPORTANCE Protein structures are more strongly conserved in evolution than are amino acid sequences. Comparative structural analysis is particularly important for inferring the origins of viral proteins that typically evolve at high rates. We used a powerful protein structure modeling method, namely, AlphaFold2, to model the structures of all orthopoxvirus proteins and compared them to all available protein structures. Multiple cases of recruitment of host enzymes for structural roles in viruses, accompanied by the disruption of catalytic sites, were discovered. However, many viral proteins appear to have evolved unique structural folds.

RNA-activated protein cleavage with a CRISPR-associated endopeptidase.

In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.

Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA.

RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies1,2. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development3. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain4. IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems.

Human pathogenic RNA viruses establish noncompeting lineages by occupying independent niches.

Many pathogenic viruses are endemic among human populations and can cause a broad variety of diseases, some potentially leading to devastating pandemics. How virus populations maintain diversity and what selective pressures drive population turnover is not thoroughly understood. We conducted a large-scale phylodynamic analysis of 27 human pathogenic RNA viruses spanning diverse life history traits, in search of unifying trends that shape virus evolution. For most virus species, we identify multiple, cocirculating lineages with low turnover rates. These lineages appear to be largely noncompeting and likely occupy semiindependent epidemiological niches that are not regionally or seasonally defined. Typically, intralineage mutational signatures are similar to interlineage signatures. The principal exception are members of the family Picornaviridae, for which mutations in capsid protein genes are primarily lineage defining. Interlineage turnover is slower than expected under a neutral model, whereas intralineage turnover is faster than the neutral expectation, further supporting the existence of independent niches. The persistence of virus lineages appears to stem from limited outbreaks within small communities, so that only a small fraction of the global susceptible population is infected at any time. As disparate communities become increasingly connected through globalization, interaction and competition between lineages might increase as well, which could result in changing selective pressures and increased diversification and/or pathogenicity. Thus, in addition to zoonotic events, ongoing surveillance of familiar, endemic viruses appears to merit global attention with respect to the prevention or mitigation of future pandemics.

Epistasis at the SARS-CoV-2 Receptor-Binding Domain Interface and the Propitiously Boring Implications for Vaccine Escape.

At the time of this writing, December 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlaps the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple RBD mutations have risen to dominance. Nonadditive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape, particularly the risk of vaccine escape, is crucial. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the wild type and Delta, Gamma, and Omicron variants. Overall, epistasis at the RBD interface appears to be limited, and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2 interaction, whereas in Gamma, epistasis more substantially destabilizes NAb interaction. Despite bearing many more RBD mutations, the epistatic landscape of Omicron closely resembles that of Gamma. Thus, although Omicron poses new risks not observed with Delta, structural constraints on the RBD appear to hamper continued evolution toward more complete vaccine escape. The modest ensemble of mutations relative to the wild type that are currently known to reduce vaccine efficacy is likely to contain the majority of all possible escape mutations for future variants, predicting the continued efficacy of the existing vaccines. IMPORTANCE Emergence of vaccine escape variants of SARS-CoV-2 is arguably the most pressing problem during the COVID-19 pandemic as vaccines are distributed worldwide. We employed a computational approach to assess the risk of antibody escape resulting from mutations in the receptor-binding domain of the spike protein of the wild-type SARS-CoV-2 virus as well as the Delta, Gamma, and Omicron variants. The efficacy of the existing vaccines against Omicron could be substantially reduced relative to the wild type, and the potential for vaccine escape is of grave concern. Our results suggest that although Omicron poses new evolutionary risks not observed for Delta, structural constraints on the RBD make continued evolution toward more complete vaccine escape from either Delta or Omicron unlikely. The modest set of escape-enhancing mutations already identified for the wild type likely include the majority of all possible mutations with this effect.

Cargo Genes of Tn7-Like Transposons Comprise an Enormous Diversity of Defense Systems, Mobile Genetic Elements, and Antibiotic Resistance Genes.

Transposition is a major mechanism of horizontal gene mobility in prokaryotes. However, exploration of the genes mobilized by transposons (cargo) is hampered by the difficulty in delineating integrated transposons from their surrounding genetic context. Here, we present a computational approach that allowed us to identify the boundaries of 6,549 Tn7-like transposons. We found that 96% of these transposons carry at least one cargo gene. Delineation of distinct communities in a gene-sharing network demonstrates how transposons function as a conduit of genes between phylogenetically distant hosts. Comparative analysis of the cargo genes reveals significant enrichment of mobile genetic elements (MGEs) nested within Tn7-like transposons, such as insertion sequences and toxin-antitoxin modules, and of genes involved in recombination, anti-MGE defense, and antibiotic resistance. More unexpectedly, cargo also includes genes encoding central carbon metabolism enzymes. Twenty-two Tn7-like transposons carry both an anti-MGE defense system and antibiotic resistance genes, illustrating how bacteria can overcome these combined pressures upon acquisition of a single transposon. This work substantially expands the distribution of Tn7-like transposons, defines their evolutionary relationships, and provides a large-scale functional classification of prokaryotic genes mobilized by transposition. IMPORTANCE Transposons are major vehicles of horizontal gene transfer that, in addition to genes directly involved in transposition, carry cargo genes. However, characterization of these genes is hampered by the difficulty of identification of transposon boundaries. We developed a computational approach for detecting transposon ends and applied it to perform a comprehensive census of the cargo genes of Tn7-like transposons, a large class of bacterial mobile genetic elements (MGE), many of which employ a unique, CRISPR-mediated mechanism of site-specific transposition. The cargo genes encompass a striking diversity of MGE, defense, and antibiotic resistance systems. Unexpectedly, we also identified cargo genes encoding metabolic enzymes. Thus, Tn7-like transposons mobilize a vast repertoire of genes that can have multiple effects on the host bacteria.

Epistasis at the SARS-CoV-2 RBD Interface and the Propitiously Boring Implications for Vaccine Escape.

At the time of this writing, December 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlap with the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple mutations in the RBD have rose to dominance. Non-additive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape is crucial. Epistasis can substantially increase the risk of vaccine escape and cannot be completely characterized through the study of the wild type (WT) alone. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the WT, Delta, Gamma, and Omicron variants. Overall, epistasis at the RBD interface appears to be limited and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2 interaction, whereas in the Gamma variant, epistasis more substantially destabilizes NAb interaction. Although a small, systematic trend towards NAb destabilization not observed for Delta or Gamma was detected for Omicron, and despite bearing significantly more RBD mutations, the epistatic landscape of the Omicron variant closely resembles that of Gamma. These results suggest that, although Omicron poses new risks not observed with Delta, structural constraints on the RBD hamper continued evolution towards more complete vaccine escape. The modest ensemble of mutations relative to the WT that are currently known to reduce vaccine efficacy is likely to comprise the majority of all possible escape mutations for future variants, predicting continued efficacy of the existing vaccines.

Ongoing global and regional adaptive evolution of SARS-CoV-2.

Understanding the trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution is paramount to control the COVID-19 pandemic. We analyzed more than 300,000 high-quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the region of the nucleocapsid protein associated with nuclear localization signals (NLS) are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region-specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS-associated variants across multiple partitions rose to global prominence in March to July, during a period of stasis in terms of interregional diversity. Finally, beginning in July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation.

Mitochondria-rough-ER contacts in the liver regulate systemic lipid homeostasis.

Contacts between organelles create microdomains that play major roles in regulating key intracellular activities and signaling pathways, but whether they also regulate systemic functions remains unknown. Here, we report the ultrastructural organization and dynamics of the inter-organellar contact established by sheets of curved rough endoplasmic reticulum closely wrapped around the mitochondria (wrappER). To elucidate the in vivo function of this contact, mouse liver fractions enriched in wrappER-associated mitochondria are analyzed by transcriptomics, proteomics, and lipidomics. The biochemical signature of the wrappER points to a role in the biogenesis of very-low-density lipoproteins (VLDL). Altering wrappER-mitochondria contacts curtails VLDL secretion and increases hepatic fatty acids, lipid droplets, and neutral lipid content. Conversely, acute liver-specific ablation of Mttp, the most upstream regulator of VLDL biogenesis, recapitulates this hepatic dyslipidemia phenotype and promotes remodeling of the wrappER-mitochondria contact. The discovery that liver wrappER-mitochondria contacts participate in VLDL biology suggests an involvement of inter-organelle contacts in systemic lipid homeostasis.

Dual modes of CRISPR-associated transposon homing.

Tn7-like transposons have co-opted CRISPR systems, including class 1 type I-F, I-B, and class 2 type V-K. Intriguingly, although these CRISPR-associated transposases (CASTs) undergo robust CRISPR RNA (crRNA)-guided transposition, they are almost never found in sites targeted by the crRNAs encoded by the cognate CRISPR array. To understand this paradox, we investigated CAST V-K and I-B systems and found two distinct modes of transposition: (1) crRNA-guided transposition and (2) CRISPR array-independent homing. We show distinct CAST systems utilize different molecular mechanisms to target their homing site. Type V-K CAST systems use a short, delocalized crRNA for RNA-guided homing, whereas type I-B CAST systems, which contain two distinct target selector proteins, use TniQ for RNA-guided DNA transposition and TnsD for homing to an attachment site. These observations illuminate a key step in the life cycle of CAST systems and highlight the diversity of molecular mechanisms mediating transposon homing.

Ongoing Global and Regional Adaptive Evolution of SARS-CoV-2.

Understanding the trends in SARS-CoV-2 evolution is paramount to control the COVID-19 pandemic. We analyzed more than 300,000 high quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the nuclear localization signal (NLS) associated region of the nucleocapsid protein are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region-specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS associated variants across multiple partitions rose to global prominence in March-July, during a period of stasis in terms of inter-regional diversity. Finally, beginning July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation.

Genomic determinants of pathogenicity in SARS-CoV-2 and other human coronaviruses.

SARS-CoV-2 poses an immediate, major threat to public health across the globe. Here we report an in-depth molecular analysis to reconstruct the evolutionary origins of the enhanced pathogenicity of SARS-CoV-2 and other coronaviruses that are severe human pathogens. Using integrated comparative genomics and machine learning techniques, we identify key genomic features that differentiate SARS-CoV-2 and the viruses behind the two previous deadly coronavirus outbreaks, SARS-CoV and MERS-CoV, from less pathogenic coronaviruses. These features include enhancement of the nuclear localization signals in the nucleocapsid protein and distinct inserts in the spike glycoprotein that appear to be associated with high case fatality rate of these coronaviruses as well as the host switch from animals to humans. The identified features could be crucial elements of coronavirus pathogenicity and possible targets for diagnostics, prognostication and interventions.

Genomic determinants of pathogenicity in SARS-CoV-2 and other human coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an immediate, major threat to public health across the globe. Here we report an in-depth molecular analysis to reconstruct the evolutionary origins of the enhanced pathogenicity of SARS-CoV-2 and other coronaviruses that are severe human pathogens. Using integrated comparative genomics and machine learning techniques, we identify key genomic features that differentiate SARS-CoV-2 and the viruses behind the two previous deadly coronavirus outbreaks, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), from less pathogenic coronaviruses. These features include enhancement of the nuclear localization signals in the nucleocapsid protein and distinct inserts in the spike glycoprotein that appear to be associated with high case fatality rate of these coronaviruses as well as the host switch from animals to humans. The identified features could be crucial contributors to coronavirus pathogenicity and possible targets for diagnostics, prognostication, and interventions.