Cut-and-Run: A Distinct Mechanism by which V(D)J Recombination Causes Genome Instability.

V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo. Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction "cut-and-run" and suggest that it could be a significant cause of lymphocyte genome instability.

A bovine antibody possessing an ultralong complementarity-determining region CDRH3 targets a highly conserved epitope in sarbecovirus spike proteins.

Broadly neutralizing antibodies have huge potential as novel antiviral therapeutics due to their ability to recognize highly conserved epitopes that are seldom mutated in viral variants. A subset of bovine antibodies possess an ultralong complementarity-determining region (CDR)H3 that is highly adept at recognizing such conserved epitopes, but their reactivity against Sarbecovirus Spike proteins has not been explored previously. Here, we use a SARS-naïve library to isolate a broadly reactive bovine CDRH3 that binds the receptor-binding domain of SARS-CoV, SARS-CoV-2, and all SARS-CoV-2 variants. We show further that it neutralizes viruses pseudo-typed with SARS-CoV Spike, but this is not by competition with angiotensin-converting enzyme 2 (ACE2) binding. Instead, using differential hydrogen-deuterium exchange mass spectrometry, we demonstrate that it recognizes the major site of vulnerability of Sarbecoviruses. This glycan-shielded cryptic epitope becomes available only transiently via interdomain movements of the Spike protein such that antibody binding triggers destruction of the prefusion complex. This proof of principle study demonstrates the power of in vitro expressed bovine antibodies with ultralong CDRH3s for the isolation of novel, broadly reactive tools to combat emerging pathogens and to identify key epitopes for vaccine development.

Is intestinal transport dysfunctional in COVID-19-related diarrhea?

Diarrhea, often severe, is a recognized and frequently early symptom during acute COVID-19 infection and may persist or develop for the first time in patients with long-COVID, with socioeconomic consequences. Diarrheal mechanisms in these cases are poorly understood. There is evidence for disruption of intestinal epithelial barrier function and also for changes in the gut microbiome, which is critical for gut immunity and metabolism. Whether the SARS-CoV-2 virus has adverse effects on intestinal transport proteins is unclear. However, the ability of the virus to inhibit expression and activity of an aldosterone-regulated epithelial sodium (Na+) channel (ENaC) present in human distal colon, which is responsible for Na+ and water salvage, points to possible disruption of other intestinal transport proteins during COVID-19 infection. In this Perspective, we develop this idea by highlighting possible intestinal transport protein targets for the SARS-CoV-2 virus and discussing how their interactions might be explored in the laboratory.

Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts.

Picornaviruses are important viral pathogens, but despite extensive study, the assembly process of their infectious virions is still incompletely understood, preventing the development of anti-viral strategies targeting this essential part of the life cycle. We report the identification, via RNA SELEX and bioinformatics, of multiple RNA sites across the genome of a typical enterovirus, enterovirus-E (EV-E), that each have affinity for the cognate viral capsid protein (CP) capsomer. Many of these sites are evolutionarily conserved across known EV-E variants, suggesting they play essential functional roles. Cryo-electron microscopy was used to reconstruct the EV-E particle at ~2.2 Å resolution, revealing extensive density for the genomic RNA. Relaxing the imposed symmetry within the reconstructed particles reveals multiple RNA-CP contacts, a first for any picornavirus. Conservative mutagenesis of the individual RNA-contacting amino acid side chains in EV-E, many of which are conserved across the enterovirus family including poliovirus, is lethal but does not interfere with replication or translation. Anti-EV-E and anti-poliovirus aptamers share sequence similarities with sites distributed across the poliovirus genome. These data are consistent with the hypothesis that these RNA-CP contacts are RNA Packaging Signals (PSs) that play vital roles in assembly and suggest that the RNA PSs are evolutionarily conserved between pathogens within the family, augmenting the current protein-only assembly paradigm for this family of viruses.

Rewriting nature's assembly manual for a ssRNA virus.

Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes.

Revealing the density of encoded functions in a viral RNA.

We present direct experimental evidence that assembly of a single-stranded RNA virus occurs via a packaging signal-mediated mechanism. We show that the sequences of coat protein recognition motifs within multiple, dispersed, putative RNA packaging signals, as well as their relative spacing within a genomic fragment, act collectively to influence the fidelity and yield of capsid self-assembly in vitro. These experiments confirm that the selective advantages for viral yield and encapsidation specificity, predicted from previous modeling of packaging signal-mediated assembly, are found in Nature. Regions of the genome that act as packaging signals also function in translational and transcriptional enhancement, as well as directly coding for the coat protein, highlighting the density of encoded functions within the viral RNA. Assembly and gene expression are therefore direct molecular competitors for different functional folds of the same RNA sequence. The strongest packaging signal in the test fragment, encodes a region of the coat protein that undergoes a conformational change upon contact with packaging signals. A similar phenomenon occurs in other RNA viruses for which packaging signals are known. These contacts hint at an even deeper density of encoded functions in viral RNA, which if confirmed, would have profound consequences for the evolution of this class of pathogens.

Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment.

Reoviruses are important human, animal and plant pathogens having 10-12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA-protein and RNA-RNA interactions between viral genomic segment precursors have been implicated in the process. While non-structural viral RNA-binding proteins, such as avian reovirus σNS, are essential for virus replication, the mechanism by which they assist packaging is unclear. Here we demonstrate that σNS assembles into stable elongated hexamers in vitro, which bind single-stranded nucleic acids with high affinity, but little sequence specificity. Using ensemble and single molecule fluorescence spectroscopy, we show that σNS also binds to a partially double-stranded RNA, resulting in gradual helix unwinding. The hexamer can bind multiple RNA molecules and exhibits strand-annealing activity, thus mediating conversion of metastable, intramolecular stem-loops into more stable heteroduplexes. We demonstrate that the ARV σNS acts as an RNA chaperone facilitating specific RNA-RNA interactions between genomic precursors during segment assortment and packaging.

Solving a Levinthal's paradox for virus assembly identifies a unique antiviral strategy.

One of the important puzzles in virology is how viruses assemble the protein containers that package their genomes rapidly and efficiently in vivo while avoiding triggering their hosts' antiviral defenses. Viral assembly appears directed toward a relatively small subset of the vast number of all possible assembly intermediates and pathways, akin to Levinthal's paradox for the folding of polypeptide chains. Using an in silico assembly model, we demonstrate that this reduction in complexity can be understood if aspects of in vivo assembly, which have mostly been neglected in in vitro experimental and theoretical modeling assembly studies, are included in the analysis. In particular, we show that the increasing viral coat protein concentration that occurs in infected cells plays unexpected and vital roles in avoiding potential kinetic assembly traps, significantly reducing the number of assembly pathways and assembly initiation sites, and resulting in enhanced assembly efficiency and genome packaging specificity. Because capsid assembly is a vital determinant of the overall fitness of a virus in the infection process, these insights have important consequences for our understanding of how selection impacts on the evolution of viral quasispecies. These results moreover suggest strategies for optimizing the production of protein nanocontainers for drug delivery and of virus-like particles for vaccination. We demonstrate here in silico that drugs targeting the specific RNA-capsid protein contacts can delay assembly, reduce viral load, and lead to an increase of misencapsidation of cellular RNAs, hence opening up unique avenues for antiviral therapy.

Sizes of Long RNA Molecules Are Determined by the Branching Patterns of Their Secondary Structures.

Long RNA molecules are at the core of gene regulation across all kingdoms of life, while also serving as genomes in RNA viruses. Few studies have addressed the basic physical properties of long single-stranded RNAs. Long RNAs with nonrepeating sequences usually adopt highly ramified secondary structures and are better described as branched polymers. To test whether a branched polymer model can estimate the overall sizes of large RNAs, we employed fluorescence correlation spectroscopy to examine the hydrodynamic radii of a broad spectrum of biologically important RNAs, ranging from viral genomes to long noncoding regulatory RNAs. The relative sizes of long RNAs measured at low ionic strength correspond well to those predicted by two theoretical approaches that treat the effective branching associated with secondary structure formation-one employing the Kramers theorem for calculating radii of gyration, and the other featuring the metric of maximum ladder distance. Upon addition of multivalent cations, most RNAs are found to be compacted as compared with their original, low ionic-strength sizes. These results suggest that sizes of long RNA molecules are determined by the branching pattern of their secondary structures. We also experimentally validate the proposed computational approaches for estimating hydrodynamic radii of single-stranded RNAs, which use generic RNA structure prediction tools and thus can be universally applied to a wide range of long RNAs.

Asymmetric genome organization in an RNA virus revealed via graph-theoretical analysis of tomographic data.

Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug design.

Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies.

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ(70) or σ(54), that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ(54) version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ(70) and σ(54), the domain movements of the latter have evolved to require an activator ATPase.

Distinguishing closely related amyloid precursors using an RNA aptamer.

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human ß2-microglobulin (hß2m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hß2m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2'F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hß2m with an EC50 of ∼200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-hß2m complex, revealing that the aptamer binds to the face of hß2m containing the A, B, E, and D ß-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hß2m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.

Limits of structural plasticity in a picornavirus capsid revealed by a massively expanded equine rhinitis A virus particle.

UNLABELLED: The Picornaviridae family of small, nonenveloped viruses includes major pathogens of humans and animals. They have positive-sense, single-stranded RNA genomes, and the mechanism(s) by which these genomes are introduced into cells to initiate infection remains poorly understood. The structures of presumed uncoating intermediate particles of several picornaviruses show limited expansion and some increased porosity compared to the mature virions. Here, we present the cryo-electron microscopy structure of native equine rhinitis A virus (ERAV), together with the structure of a massively expanded ERAV particle, each at ∼17-Šresolution. The expanded structure has large pores on the particle 3-fold axes and has lost the RNA genome and the capsid protein VP4. The expanded structure thus illustrates both the limits of structural plasticity in such capsids and a plausible route by which genomic RNA might exit. IMPORTANCE: Picornaviruses are important animal and human pathogens that protect their genomic RNAs within a protective protein capsid. Upon infection, this genomic RNA must be able to leave the capsid to initiate a new round of infection. We describe here the structure of a unique, massively expanded state of equine rhinitis A virus that provides insight into how this exit might occur.

Oncogene dependency and the potential of targeted RNAi-based anti-cancer therapy.

Cancers arise through the progression of multiple genetic and epigenetic defects that lead to deregulation of numerous signalling networks. However, the last decade has seen the development of the concept of 'oncogene addiction', where tumours appear to depend on a single oncogene for survival. RNAi has provided an invaluable tool in the identification of these oncogenes and oncogene-dependent cancers, and also presents great potential as a novel therapeutic strategy against them. Although RNAi therapeutics have demonstrated effective killing of oncogene-dependent cancers in vitro, their efficacy in vivo is severely limited by effective delivery systems. Several virus-based RNAi delivery strategies have been explored, but problems arose associated with high immunogenicity, random genome integration and non-specific targeting. This has directed efforts towards non-viral formulations, including delivery systems based on virus-like particles, liposomes and cationic polymers, which can circumvent some of these problems by immunomasking and the use of specific tumour-targeting ligands. This review outlines the prevalence of oncogene-dependent cancers, evaluates the potential of RNAi-based therapeutics and assesses the relative strengths and weaknesses of different approaches to targeted RNAi delivery.

Evidence that viral RNAs have evolved for efficient, two-stage packaging.

Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from differing structural families, we have discovered that packaging is a two-stage process. Initially, the genomic RNAs undergo rapid and dramatic (approximately 20-30%) collapse of their solution conformations upon addition of cognate coat proteins. The collapse occurs with a substoichiometric ratio of coat protein subunits and is followed by a gradual increase in particle size, consistent with the recruitment of additional subunits to complete a growing capsid. Equivalently sized nonviral RNAs, including high copy potential in vivo competitor mRNAs, do not collapse. They do support particle assembly, however, but yield many aberrant structures in contrast to viral RNAs that make only capsids of the correct size. The collapse is specific to viral RNA fragments, implying that it depends on a series of specific RNA-protein interactions. For bacteriophage MS2, we have shown that collapse is driven by subsequent protein-protein interactions, consistent with the RNA-protein contacts occurring in defined spatial locations. Conformational collapse appears to be a distinct feature of viral RNA that has evolved to facilitate assembly. Aspects of this process mimic those seen in ribosome assembly.

The influence of two-dimensional organization on peptide conformation.

Molecular crowding plays a significant role in regulating molecular conformation in cellular environments. It is also likely to be important wherever high molecular densities are required, for example in surface-phase studies, in which molecular densities generally far exceed those observed in solution. Using on-surface circular dichroism (CD) spectroscopy, we have investigated the structure of a synthetic peptide assembled into a highly packed monolayer. The immobilized peptide undergoes a structural transition between α-helical and random coil conformation upon changes in pH and ionic concentration, but critically the threshold for conformational change is altered dramatically by molecular crowding within the peptide monolayer. This study highlights the often overlooked role molecular crowding plays in regulating molecular structure and function in surface-phase studies of biological molecules.

Expanding the repertoire of amyloid polymorphs by co-polymerization of related protein precursors.

Amyloid fibrils can be generated from proteins with diverse sequences and folds. Although amyloid fibrils assembled in vitro commonly involve a single protein precursor, fibrils formed in vivo can contain more than one protein sequence. How fibril structure and stability differ in fibrils composed of single proteins (homopolymeric fibrils) from those generated by co-polymerization of more than one protein sequence (heteropolymeric fibrils) is poorly understood. Here we compare the structure and stability of homo and heteropolymeric fibrils formed from human ß2-microglobulin and its truncated variant ΔN6. We use an array of approaches (limited proteolysis, magic angle spinning NMR, Fourier transform infrared spectroscopy, and fluorescence) combined with measurements of thermodynamic stability to characterize the different fibril types. The results reveal fibrils with different structural properties, different side-chain packing, and strikingly different stabilities. These findings demonstrate how co-polymerization of related precursor sequences can expand the repertoire of structural and thermodynamic polymorphism in amyloid fibrils to an extent that is greater than that obtained by polymerization of a single precursor alone.

Molecular frustration: a hypothesis for regulation of viral infections.

The recent revolution in imaging techniques and results from RNA footprinting in situ reveal how the bacteriophage MS2 genome regulates both particle assembly and genome release. We have proposed a model in which multiple packaging signal (PS) RNA-coat protein (CP) contacts orchestrate different stages of a viral life cycle. Programmed formation and release of specific PS contacts with CP regulates viral particle assembly and genome uncoating during cell entry. We hypothesize that molecular frustration, a concept introduced to understand protein folding, can be used to better rationalize how PSs function in both particle assembly and genome release. More broadly this concept may explain the directionality of viral life cycles, for example, the roles of host cofactors in HIV infection. We propose that this is a universal principle in virology that explains mechanisms of host-virus interaction and suggests diverse therapeutic interventions.

MS2 viruslike particles: a robust, semisynthetic targeted drug delivery platform.

We show that viruslike particles (VLPs) reassembled in vitro with the RNA bacteriophage MS2 coat protein and an RNA conjugate encompassing a siRNA and a known capsid assembly signal can be targeted to HeLa cells by covalent attachment of human transferrin. The siRNA VLPs protect their cargoes from nuclease, have a double-stranded conformation in the capsid and carry multiple drug and targeting ligands. The relative efficiency of VLP reassembly has been assessed, and conditions have been determined for larger scale production. Targeted VLPs have been purified away from unmodified VLPs for the first time allowing improved analysis of the effects of this synthetic virion system. The particles enter cells via receptor-mediated endocytosis and produce siRNA effects at low nanomolar concentrations. Although less effective than a commercial cationic lipid vector at siRNA delivery, the smaller amounts of internalized RNA with VLP delivery had an effect as good as if not better than the lipid transfection route. This implies that the siRNAs delivered by this route are more accessible to the siRNA pathway than identical RNAs delivered in complex lipid aggregates. The data suggest that the MS2 system continues to show many of the features that will be required to create an effective targeted drug delivery system. The fluorescence assays of siRNA effects described here will facilitate the combinatorial analysis of both future formulations and dosing regimes.

A two-stage mechanism of viral RNA compaction revealed by single molecule fluorescence.

Long RNAs often exist as multiple conformers in equilibrium. For the genomes of single-stranded RNA viruses, one of these conformers must include a compacted state allowing the RNA to be confined within the virion. We have used single molecule fluorescence correlation spectroscopy to monitor the conformations of viral genomes and sub-fragments in the absence and presence of coat proteins. Cognate RNA-coat protein interactions in two model viruses cause a rapid collapse in the hydrodynamic radii of their respective RNAs. This is caused by protein binding at multiple sites on the RNA that facilitate additional protein-protein contacts. The collapsed species recruit further coat proteins to complete capsid assembly with great efficiency and fidelity. The specificity in RNA-coat protein interactions seen at single-molecule concentrations reflects the packaging selectivity seen for such viruses in vivo. This contrasts with many in vitro reassembly measurements performed at much higher concentrations. RNA compaction by coat protein or polycation binding are distinct processes, implying that defined RNA-coat protein contacts are required for assembly.