Efficient functioning of the prokaryotic translational system depends on a steady supply of aminoacylated tRNAs to be delivered to translating ribosomes via ternary complex. As such, tRNA synthetases play a crucial role in maintaining efficient and accurate translation in the cell, as they are responsible for aminoacylating the correct amino acid to its corresponding tRNA. Moreover, the kinetic rate at which they perform this reaction will dictate the overall rate of supply of aminoacylated tRNAs to the ribosome and will have consequences for the average translational speed of ribosomes in the cell. In this work, I develop an empirical kinetic model for the 20 aminoacyl tRNA synthetase enzymes in E. coli enabling the study of the effects of tRNA charging dynamics on translational efficiency. The model is parametrised based on in vitro experimental measurements of substrate Km and kcat values for both pyrophosphate exchange and aminoacylation. The model also reproduces the burst kinetics observed in class I enzymes and the transfer rates measured in single turnover experiments. Stochastic simulation of in vivo translation shows the kinetic model is able to support the tRNA charging demand resulting from translation in exponentially growing E. coli cells at a variety of different doubling times. This work provides a basis for the theoretical study of the amino acid starvation and the stringent response, as well as the complex behaviour of tRNA charging and translational dynamics in response to cellular stresses.

Protein expression in vivo is predominately controlled via regulatory feedback mechanisms that adjust the level of mRNA transcription. However for positive sense single-stranded RNA viruses, protein expression is often controlled via secondary structural elements, such as internal ribosomal entry sites, that are encoded within the mRNA. The self-regulation of mRNA translation observed in this class of viruses suggests that it may be possible to design mRNAs that self-regulate their protein expression, enabling the creation of mRNAs for vaccines and other synthetic biology applications where protein levels in the cell can be tightly controlled without feedback to a transcriptional mechanism. As a proof of concept, I design a polycistronic mRNA based on bacteriophage MS2, where the upstream gene is capable of repressing synthesis of the downstream gene. Using a computational tool that simulates ribosome kinetics and the co-translational folding of the mRNA in response, I show that mutations to the mRNA can be identified which enhance the efficiency of the translation and the repression of the downstream gene. The results of this study open up the possibility of designing bespoke mRNA gene circuits in which the amount of protein synthesised in cells are self-regulated for therapeutic or antigenic purposes.

The control of protein synthesis and the overall levels of various proteins in the cell is critical for achieving homoeostasis. Regulation of protein levels can occur at the transcriptional level, where the total number of messenger RNAs in the overall transcriptome are controlled, or at the translational level, where interactions of proteins and ribosomes with the messenger RNA determine protein translational efficiency. Although transcriptional control of mRNA levels is the most commonly used regulatory control mechanism in cells, positive-sense single-stranded RNA viruses often utilise translational control mechanisms to regulate their proteins in the host cell. Here I detail a computational method for stochastically simulating protein synthesis on a dynamic messenger RNA using the Gillespie algorithm, where the mRNA is allowed to co-translationally fold in response to ribosome movement. Applying the model to the test case of the bacteriophage MS2 virus, I show that the models ability to accurately reproduce experimental measurements of coat protein production and translational repression of the viral RNA dependant RNA polymerase at high coat protein concentrations. The computational techniques reported here open up the potential to examine the infection dynamics of a ssRNA virus in a host cell at the level of the genomic RNA, as well as examine general translation control mechanisms present in polycistronic mRNAs.

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 tran-scripts, 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 tran-siently 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 30 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. (C) 2022 The Authors. Published by Elsevier Ltd.

The vast majority of viruses consist of a nucleic acid surrounded by a protective icosahedral protein shell called the capsid. During viral infection of a host cell, the timing and efficiency of the assembly process is important for ensuring the production of infectious new progeny virus particles. In the class of single-stranded RNA (ssRNA) viruses, the assembly of the capsid takes place in tandem with packaging of the ssRNA genome in a highly cooperative co-assembly process. In simple ssRNA viruses such as the bacteriophage MS2 and small RNA plant viruses such as STNV, this cooperative process results from multiple interactions between the protein shell and sites in the RNA genome which have been termed packaging signals. Using a stochastic assembly algorithm which includes cooperative interactions between the protein shell and packaging signals in the RNA genome, we demonstrate that highly efficient assembly of STNV capsids arises from a set of simple local rules. Altering the local assembly rules results in different nucleation scenarios with varying assembly efficiencies, which in some cases depend strongly on interactions with RNA packaging signals. Our results provide a potential simple explanation based on local assembly rules for the ability of some ssRNA viruses to spontaneously assemble around charged polymers and other non-viral RNAs in vitro.

Within-host models of COVID-19 infection dynamics enable the merits of different forms of antiviral therapy to be assessed in individual patients. A stochastic agent-based model of COVID-19 intracellular dynamics is introduced here, that incorporates essential steps of the viral life cycle targeted by treatment options. Integration of model predictions with an intercellular ODE model of within-host infection dynamics, fitted to patient data, generates a generic profile of disease progression in patients that have recovered in the absence of treatment. This is contrasted with the profiles obtained after variation of model parameters pertinent to the immune response, such as effector cell and antibody proliferation rates, mimicking disease progression in immunocompromised patients. These profiles are then compared with disease progression in the presence of antiviral and convalescent plasma therapy against COVID-19 infections. The model reveals that using both therapies in combination can be very effective in reducing the length of infection, but these synergistic effects decline with a delayed treatment start. Conversely, early treatment with either therapy alone can actually increase the duration of infection, with infectious virions still present after the decline of other markers of infection. This suggests that usage of these treatments should remain carefully controlled in a clinical environment.

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.

The secondary structures of the bacteriophage MS2 ssRNA genome, frozen in defined states, were determined with minimal perturbation using constraints from X-ray synchrotron footprinting (XRF). The footprints of the gRNA in the virion and as transcript are consistent with single, dominant but distinct conformations, and reveal the presence of multiple Packaging Signals potentially involved in assembly regulation that have not been detected by other techniques. XRF also reveals the dramatic effect of the unique Maturation Protein (MP) on both the capsid lattice, and the gRNA conformation inside the phage compared with a virus-like-particle composed only of coat protein subunits. Aspects of genome organisation in the phage, their impacts on the capsid shell, and the distortion of lattice geometry by MP, are hallmarks of molecular frustration. Phage assembly therefore appears to prepare the particle for the next step of the infectious cycle.

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.

Recent developments in cryo-electron microscopy (cryo-EM) hardware along with continuously evolving software tools have led to the discovery of many novel structures that it was not possible to solve until now, resulting in what is termed “the resolution revolution”. In structural virology, it has also led to a re-evaluation of known structures. Most virion structures solved by X-ray crystallography or cryo-EM are focused on the capsid protein (CP) as a result of the application of icosahedral symmetry averaging to “improve” the electron density maps. However, this has the consequence that the intrinsic asymmetry of important components of virions, such as the viral genome and structural proteins lacking such symmetry, are masked. Single-stranded (ss), positive-sense RNA viruses are major pathogens in all kingdoms of life. Asymmetric cryo-EM structure determination of a major model virus in this class, bacteriophage MS2, reveals the limitations of a symmetrized view. As well as the presence and interactions made by the unique Maturation Protein, it also reveals multiple gRNA-CP dimer contacts corresponding to our previous prediction that dispersed, sequence-degenerate RNA motifs (Packaging Signals, PSs) play important roles during the virion assembly. Here, we describe how relaxing symmetry during structure determination can image such gRNA-PS contacts in a range of ssRNA viruses including the picornavirus Bovine Enterovirus-1, the alphaviruses Sindbis and Semliki Forest Viruses, as well as the plant virus Turnip Crinkle Virus. The revelation of these functionally important gRNA-CP contacts changes our fundamental understanding of assembly in these pathogens and may have further translational importance.

Computational modelling of in vivo protein synthesis is highly complicated, as it requires the simulation of ribosomal movement over the entire transcriptome, as well as consideration of the concentration effects from 40+ different types of tRNAs and numerous other protein factors. Here I report on the development of a stochastic model for protein translation that is capable of simulating the dynamical process of in vivo protein synthesis in a prokaryotic cell containing several thousand unique mRNA sequences, with explicit nucleotide information for each, and report on a number of biological predictions which are beyond the scope of existing models. In particular, I show that, when the complex network of concentration dependent interactions between elongation factors, tRNAs, ribosomes, and other factors required for protein synthesis are included in full detail, several biological phenomena, such as the increasing peptide elongation rate with bacterial growth rate, are predicted as emergent properties of the model. The stochastic model presented here demonstrates the importance of considering the translational process at this level of detail, and provides a platform to interrogate various aspects of translation that are difficult to study in more coarse-grained models.

Many single-stranded RNA viruses, including major viral pathogens, present RNA-encoded virus assembly instructions (VAIs) within their genetic message that can be isolated from the genetic code and repurposed for the design of virus-like particles. These VAIs rely on multiple dispersed RNA secondary structure elements with a consensus recognition motif for the capsid (core) protein, called packaging signals (PSs), which collectively promote capsid assembly. In this talk, I will provide evidence for the evolutionary conservation of the PS-encoded assembly instructions among different viruses in a viral family and discuss the implications of this discovery for viral evolution. I will then demonstrate how the VAIs can be exploited for therapy. In particular, defective interfering particles occur spontaneously in viral evolution as mutant strains lacking essential parts of the viral genome. Their ability to replicate in the presence of wild-type virus at the expense of virally produced resources can be mimicked by therapeutic interfering particles (TIPs). I will introduce a novel approach to the design of such TIPs based on synthetic nucleic acid sequences containing the VAIs but otherwise lacking genetic information. Using multiscale models of a viral infection, I will demonstrate the potential of these particles in both the prophylaxis and treatment of RNA viral infections.

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus . Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.

Genome segmentation offers certain evolutionary benefits to a number of pathogenic RNA viruses, including rotaviruses and influenza viruses. However, as the number of RNA segments per virion increases, the task of a non-random selection of a full set of distinct genomic RNAs poses a formidable challenge to maintaining the integrity of segmented genomes. Recently we have identified sequence-specific inter-segment interactions between rotavirus (+)ssRNA genome segment precursors. We have shown that binding of the rotavirus-encoded non-structural protein NSP2 to viral ssRNAs results in the remodeling of RNA, which is conducive to formation of inter-segment contacts. These protein-RNA interactions result in the stabilisation of extended intermolecular RNA-RNA contacts, potentially underpinning transient inter-segment interactions prior to genome encapsidation and replication. Using this approach, we have identified a number of RNA-RNA interaction sites in the rotavirus genome, which are likely to be involved in genome segment assortment process. Having established the role of NSP2 in promoting inter-segment RNA-RNA contacts, we have developed multiplexed imaging tools for directvisualization of the RNA assortment process in rotavirus-infected cells by employing single-molecule RNA FISH. To unravel the mechanisms, by which NSP2 controls the formation of inter-molecular RNA helices, we have applied RNA structure probing methods that allowed us to monitor conformational rearrangements, which are prerequisite for theformation of the RNA assortment complex. Our findings open up unique avenues for understanding the challenges for further improvement of the recently developed fully plasmid-based reverse genetics systems for rotaviruses.

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus. Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-A and 4.4-A resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-A resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.

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.

Previously, a stochastic model of single-stranded RNA virus assembly was created to model the cooperative effects between capsid proteins and genomic RNA that would occur in a packaging signal-mediated assembly process. In such an assembly scenario, multiple secondary structural elements from within the RNA, termed "packaging signals" (PS), contact coat proteins and facilitate efficient capsid assembly. In this work, the assembly model is extended to incorporate explicit nucleotide sequence information as well as simple aspects of RNA folding that would be occurring during the RNA/capsid coassembly process. Applying this paradigm to a dodecahedral viral capsid, a computer-derived nucleotide sequence is evolved de novo that is optimal for packaging the RNA into capsids, while also containing capacity for coding for a viral protein. Analysis of the effects of mutations on the ability of the RNA sequence to successfully package into a viral capsid reveals a complex fitness landscape where the majority of mutations are neutral with respect to packaging efficiency with a small number of mutations resulting in a near-complete loss of RNA packaging. Moreover, the model shows how attempts to ablate PSs in the viral RNA sequence may result in redundant PSs already present in the genome fulfilling their packaging role. This explains why recent experiments that attempt to ablate putative PSs may not see an effect on packaging. This modeling framework presents an example of how an implicit mapping can be made from genotype to a fitness parameter important for viral biology, i.e., viral capsid yield, with potential applications to theoretical models of viral evolution.

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. Sequence-specific interactions between the Hepatitis B virus pre-genome and the core protein define the nucleocapsid assembly pathway and trigger virus-like particle formation. Cp-RNA contacts may regulate pre-genome organization, facilitating reverse transcription.