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.

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.

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.

Genome-regulated Assembly of a ssRNA Virus May Also Prepare It for Infection.

Many single-stranded, positive-sense RNA viruses regulate assembly of their infectious virions by forming multiple, cognate coat protein (CP)-genome contacts at sites termed Packaging Signals (PSs). We have determined the secondary structures of the bacteriophage MS2 ssRNA genome (gRNA) frozen in defined states using constraints from X-ray synchrotron footprinting (XRF). Comparison of the footprints from phage and transcript confirms the presence of multiple PSs in contact with CP dimers in the former. This is also true for a virus-like particle (VLP) assembled around the gRNA in vitro in the absence of the single-copy Maturation Protein (MP) found in phage. Since PS folds are present at many sites across gRNA transcripts, it appears that this genome has evolved to facilitate this mechanism of assembly regulation. There are striking differences between the gRNA-CP contacts seen in phage and the VLP, suggesting that the latter are inappropriate surrogates for aspects of phage structure/function. Roughly 50% of potential PS sites in the gRNA are not in contact with the protein shell of phage. However, many of these sit adjacent to, albeit not in contact with, PS-binding sites on CP dimers. We hypothesize that these act as PSs transiently during assembly but subsequently dissociate. Combining the XRF data with PS locations from an asymmetric cryo-EM reconstruction suggests that the genome positions of such dissociations are non-random and may facilitate infection. The loss of many PS-CP interactions towards the 3' end of the gRNA would allow this part of the genome to transit more easily through the narrow basal body of the pilus extruding machinery. This is the known first step in phage infection. In addition, each PS-CP dissociation event leaves the protein partner trapped in a non-lowest free-energy conformation. This destabilizes the protein shell which must disassemble during infection, further facilitating this stage of the life-cycle.

Dataset of high-throughput ligand screening against the RNA Packaging Signals regulating Hepatitis B Virus nucleocapsid formation.

Multiple ssRNA viruses which infect bacteria, plants or humans use RNA Packaging Signal (PS)-mediated regulation during assembly to package their genomes faithfully and efficiently. PSs typically comprise short nucleotide recognition motifs, most often presented in the unpaired region of RNA stem-loops, and often bind their cognate coat proteins (CPs) with nanomolar affinity. PSs identified to date are resilient in the face of the typical error prone replication of their virus-coded polymerases, making them potential drug targets. An immobilised array of small molecular weight, drug-like compounds was panned against a fluorescently-labelled oligonucleotide encompassing the most conserved Hepatitis B Virus (HBV) PS, PS1, known to be a major determinant in nucleocapsid formation. This identified > 70 compounds that bind PS1 uniquely in the array. The commercially available 66 of these were tested for their potential effect(s) on HBV nucleocapsid-like particle (NCP) assembly in vitro, which identified potent assembly inhibitors. Here, we describe a high-throughput screen for such effects using employing fluorescence anisotropy in a 96-well microplate format. HBV genomic RNAs (gRNA) and short oligonucleotides encompassing PS1 were 5' labelled with an Alexa Fluor 488 dye. Excess (with respect to stoichiometric T = 4 NCP formation) HBV core protein (Cp) dimers were titrated robotically into solutions containing each of these RNAs stepwise, using a Biomek 4000 liquid handling robot. The anisotropy values of these mixtures were monitored using a POLARstar microplate reader. NCP-like structures were challenged with RNase A to identify reactions that did not result in complete NCP formation. The results imply that ∼50% of the compounds prevent complete NCP formation, highlighting both PS-meditated assembly and the PS-binding compounds as potential directly-acting anti-virals with a novel molecular target. Importantly, this method allows high-throughput in vitro screening for assembly inhibitors in this major human pathogen.

Dysregulation of Hepatitis B Virus Nucleocapsid Assembly in vitro by RNA-binding Small Ligands.

RNA sequences/motifs dispersed across the genome of Hepatitis B Virus regulate formation of nucleocapsid-like particles (NCPs) by core protein (Cp) in vitro, in an epsilon/polymerase-independent fashion. These multiple RNA Packaging Signals (PSs) can each form stem-loops encompassing a Cp-recognition motif, -RGAG-, in their loops. Drug-like molecules that bind the most important of these PS sites for NCP assembly regulation with nanomolar affinities, were identified by screening an immobilized ligand library with a fluorescently-labelled, RNA oligonucleotide encompassing this sequence. Sixty-six of these "hits", with affinities ranging from low nanomolar to high micromolar, were purchased as non-immobilized versions. Their affinities for PSs and effects on NCP assembly were determined in vitro by Surface Plasmon Resonance. High-affinity ligand binding is dependent on the presence of an -RGAG- motif within the loop of the PS, consistent with ligand cross-binding between PS sites. Simple structure-activity relationships show that it is also dependent on the presence of specific functional groups in these ligands. Some compounds are potent inhibitors of in vitro NCP assembly at nanomolar concentrations. Despite appropriate logP values, these ligands do not inhibit HBV replication in cell culture. However, modelling confirms the potential of using PS-binding ligands to target NCP assembly as a novel anti-viral strategy. This also allows for computational exploration of potential synergic effects between anti-viral ligands directed at distinct molecular targets in vivo. HBV PS-regulated assembly can be dysregulated by novel small molecule RNA-binding ligands opening a novel target for developing directly-acting anti-virals against this major pathogen.

An age-structured model of hepatitis B viral infection highlights the potential of different therapeutic strategies.

Hepatitis B virus (HBV) is a global health threat, and its elimination by 2030 has been prioritised by the World Health Organisation. Here we present an age-structured model for the immune response to an HBV infection, which takes into account contributions from both cell-mediated and humoral immunity. The model has been validated using published patient data recorded during acute infection. It has been adapted to the scenarios of chronic infection, clearance of infection, and flare-ups via variation of the immune response parameters. The impacts of immune response exhaustion and non-infectious subviral particles on the immune response dynamics are analysed. A comparison of different treatment options in the context of this model reveals that drugs targeting aspects of the viral life cycle are more effective than exhaustion therapy, a form of therapy mitigating immune response exhaustion. Our results suggest that antiviral treatment is best started when viral load is declining rather than in a flare-up. The model suggests that a fast antibody production rate always leads to viral clearance, highlighting the promise of antibody therapies currently in clinical trials.

Therapeutic interfering particles exploiting viral replication and assembly mechanisms show promising performance: a modelling study.

Defective interfering particles arise spontaneously during a viral infection as mutants lacking essential parts of the viral genome. Their ability to replicate in the presence of the wild-type (WT) virus (at the expense of viable viral particles) is mimicked and exploited by therapeutic interfering particles. We propose a strategy for the design of therapeutic interfering RNAs (tiRNAs) against positive-sense single-stranded RNA viruses that assemble via packaging signal-mediated assembly. These tiRNAs contain both an optimised version of the virus assembly manual that is encoded by multiple dispersed RNA packaging signals and a replication signal for viral polymerase, but lack any protein coding information. We use an intracellular model for hepatitis C viral (HCV) infection that captures key aspects of the competition dynamics between tiRNAs and viral genomes for virally produced capsid protein and polymerase. We show that only a small increase in the assembly and replication efficiency of the tiRNAs compared with WT virus is required in order to achieve a treatment efficacy greater than 99%. This demonstrates that the proposed tiRNA design could be a promising treatment option for RNA viral infections.

In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus.

The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T = 4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry relaxation followed by asymmetric reconstruction reveal that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the ε stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.

Evolution of a virus-like architecture and packaging mechanism in a repurposed bacterial protein.

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme that lacks affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding messenger RNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid that is impermeable to nucleases, and emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing nonviral carriers for diverse vaccine and delivery applications.

An Intracellular Model of Hepatitis B Viral Infection: An In Silico Platform for Comparing Therapeutic Strategies.

Hepatitis B virus (HBV) is a major focus of antiviral research worldwide. The International Coalition to Eliminate HBV, together with the World Health Organisation (WHO), have prioritised the search for a cure, with the goal of eliminating deaths from viral hepatitis by 2030. We present here a comprehensive model of intracellular HBV infection dynamics that includes all molecular processes currently targeted by drugs and agrees well with the observed outcomes of several clinical studies. The model reveals previously unsuspected kinetic behaviour in the formation of sub-viral particles, which could lead to a better understanding of the immune responses to infection. It also enables rapid comparative assessment of the impact of different treatment options and their potential synergies as combination therapies. A comparison of available and currently developed treatment options reveals that combinations of multiple capsid assembly inhibitors perform best.

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.

Broadly Neutralizing Bovine Antibodies: Highly Effective New Tools against Evasive Pathogens?

Potent antibody-mediated neutralization is critical for an organism to combat the vast array of pathogens it will face during its lifetime. Due to the potential genetic diversity of some viruses, such as HIV-1 and influenza, standard neutralizing antibodies are often ineffective or easily evaded as their targets are masked or rapidly mutated. This has thwarted efforts to both prevent and treat HIV-1 infections and means that entirely new formulations are required to vaccinate against influenza each year. However, some rare antibodies isolated from infected individuals confer broad and potent neutralization. A subset of these broadly neutralizing antibodies possesses a long complementarity-determining 3 region of the immunoglobulin heavy chain (CDR H3). This feature generates unique antigen binding site configurations that can engage conserved but otherwise inaccessible epitope targets thus neutralizing many viral variants. Remarkably, ultralong CDR H3s are a common feature of the cow antibody repertoire and are encoded by a single variable, diversity, joining (VDJ) recombination that is extensively diversified prior to antigen exposure. Recently, it was shown that cows rapidly generate a broadly neutralizing response upon exposure to HIV-1 and this is primarily mediated by these novel ultralong antibody types. This review summarises the current knowledge of these unusual CDR H3 structures and discusses their known and potential future uses.

RNA-Mediated Virus Assembly: Mechanisms and Consequences for Viral Evolution and Therapy.

Viruses, entities composed of nucleic acids, proteins, and in some cases lipids lack the ability to replicate outside their target cells. Their components self-assemble at the nanoscale with exquisite precision-a key to their biological success in infection. Recent advances in structure determination and the development of biophysical tools such as single-molecule spectroscopy and noncovalent mass spectrometry allow unprecedented access to the detailed assembly mechanisms of simple virions. Coupling these techniques with mathematical modeling and bioinformatics has uncovered a previously unsuspected role for genomic RNA in regulating formation of viral capsids, revealing multiple, dispersed RNA sequence/structure motifs [packaging signals (PSs)] that bind cognate coat proteins cooperatively. The PS ensemble controls assembly efficiency and accounts for the packaging specificity seen in vivo. The precise modes of action of the PSs vary between viral families, but this common principle applies across many viral families, including major human pathogens. These insights open up the opportunity to block or repurpose PS function in assembly for both novel antiviral therapy and gene/drug/vaccine applications.

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 modelling paradigm for RNA virus assembly.

Virus assembly, a key stage in any viral life cycle, had long been considered to be primarily driven by protein-protein interactions and nonspecific interactions between genomic RNA and capsid protein. We review here a modelling paradigm for RNA virus assembly that illustrates the crucial roles of multiple dispersed, specific interactions between viral genomes and coat proteins in capsid assembly. The model reveals how multiple sequence-structure motifs in the genomic RNA, termed packaging signals, with a shared coat protein recognition motif enable viruses to overcome a viral assembly-equivalent of Levinthal's Paradox in protein folding. The fitness advantages conferred by this mechanism suggest that it should be widespread in viruses, opening up new perspectives on viral evolution and anti-viral therapy.

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.

Erratum: Genomic RNA folding mediates assembly of human parechovirus.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

HBV RNA pre-genome encodes specific motifs that mediate interactions with the viral core protein that promote nucleocapsid assembly.

Formation of the hepatitis B virus nucleocapsid is an essential step in the viral lifecycle, but its assembly is not fully understood. We report the discovery of sequence-specific interactions between the viral pre-genome and the hepatitis B core protein that play roles in defining the nucleocapsid assembly pathway. Using RNA SELEX and bioinformatics, we identified multiple regions in the pre-genomic RNA with high affinity for core protein dimers. These RNAs form stem-loops with a conserved loop motif that trigger sequence-specific assembly of virus-like particles (VLPs) at much higher fidelity and yield than in the absence of RNA. The RNA oligos do not interact with preformed RNA-free VLPs, so their effects must occur during particle assembly. Asymmetric cryo-electron microscopy reconstruction of the T = 4 VLPs assembled in the presence of one of the RNAs reveals a unique internal feature connected to the main core protein shell via lobes of density. Biophysical assays suggest that this is a complex involving several RNA oligos interacting with the C-terminal arginine-rich domains of core protein. These core protein-RNA contacts may play one or more roles in regulating the organization of the pre-genome during nucleocapsid assembly, facilitating subsequent reverse transcription and acting as a nucleation complex for nucleocapsid assembly.