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

Characterization of RNA aptamers that disrupt the RUNX1-CBFbeta/DNA complex.

The transcription factor RUNX1 (AML1) is an important regulator of haematopoiesis, and an important fusion partner in leukaemic translocations. High-affinity DNA binding by RUNX1 requires the interaction of the RUNX1 Runt-Homology-Domain (RHD) with the core-binding factor beta protein (CBFbeta). To generate novel reagents for in vitro and in vivo studies of RUNX1 function, we have selected high-affinity RNA aptamers against a recombinant RHD-CBFbeta complex. Selection yielded two sequence families, each dominated by a single consensus sequence. Aptamers from each family disrupt DNA binding by the RUNX1 protein in vitro and compete with sequence-specific dsDNA binding. Minimal, high-affinity ( approximately 100-160 nM) active aptamer fragments 28 and 30 nts in length, consisting of simple short stem-loop structures, were then identified. These bind to the RHD subunit and disrupt its interaction with CBFbeta, which is consistent with reduced DNA affinity in the presence of aptamer. These aptamers represent new reagents that target a novel surface on the RHD required to stabilize the recombinant RHD-CBFbeta complex and thus will further aid exploring the functions of this key transcription factor

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

Genomic RNA folding mediates assembly of human parechovirus

Assembly of the major viral pathogens of the Picornaviridae family is poorly understood. Human parechovirus 1 is an example of such viruses that contains 60 short regions of ordered RNA density making identical contacts with the protein shell. We show here via a combination of RNA-based systematic evolution of ligands by exponential enrichment, bioinformatics analysis and reverse genetics that these RNA segments are bound to the coat proteins in a sequence-specific manner. Disruption of either the RNA coat protein recognition motif or its contact amino acid residues is deleterious for viral assembly. The data are consistent with RNA packaging signals playing essential roles in virion assembly. Their binding sites on the coat proteins are evolutionarily conserved across the Parechovirus genus, suggesting that they represent potential broad-spectrum anti-viral targets.Peer reviewe

Isolation of an Asymmetric RNA Uncoating Intermediate for a Single-Stranded RNA Plant Virus

AbstractWe have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity

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 contains a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP binding motif -A.X.X.A- 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 –A.X.X.A- motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127nt 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 viruslike particles encapsidating non-native RNAs or other cargoes

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

Conformational flexibility and molecular interactions of an archaeal homologue of the Shwachman-Bodian-Diamond syndrome protein

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Defects in the human Shwachman-Bodian-Diamond syndrome (SBDS) protein-coding gene lead to the autosomal recessive disorder characterised by bone marrow dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. This protein is highly conserved in eukaryotes and archaea but is not found in bacteria. Although genomic and biophysical studies have suggested involvement of this protein in RNA metabolism and in ribosome biogenesis, its interacting partners remain largely unknown. Results We determined the crystal structure of the SBDS orthologue from Methanothermobacter thermautotrophicus (mthSBDS). This structure shows that SBDS proteins are highly flexible, with the N-terminal FYSH domain and the C-terminal ferredoxin-like domain capable of undergoing substantial rotational adjustments with respect to the central domain. Affinity chromatography identified several proteins from the large ribosomal subunit as possible interacting partners of mthSBDS. Moreover, SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments, combined with electrophoretic mobility shift assays (EMSA) suggest that mthSBDS does not interact with RNA molecules in a sequence specific manner. Conclusion It is suggested that functional interactions of SBDS proteins with their partners could be facilitated by rotational adjustments of the N-terminal and the C-terminal domains with respect to the central domain. Examination of the SBDS protein structure and domain movements together with its possible interaction with large ribosomal subunit proteins suggest that these proteins could participate in ribosome function.Published versio

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

Revealing the density of encoded functions in a viral RNA

Nikesh Patel, et al, ‘Revealing the density of encoded functions in a viral RNA’, Proceedings of the National Academy of Sciences of the United States of America (PNAS), Vol. 112 (7): 2227-2232, February 2015, doi: http:dx.doi.org/10. 1073/pnas.1420812112. This article is freely available online through the PNAS open access option.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 pathogensPeer reviewedFinal Published versio