Biology subjects

Brierley, I.

Publications and source records attributed to Brierley, I..

9 recordsLinked to original sources

bioRxiv2025

A new protein-dependent riboswitch activates ribosomal frameshifting

Programmed -1 ribosomal frameshifting (PRF) is a translational control mechanism used by RNA viruses to regulate the relative abundance of proteins encoded in different reading frames. Cardioviruses exhibit the highest known PRF efficiency, with ~85% of ribosomes shifting into the -1 frame. This unusual event requires an interaction between the viral 2A protein and a stimulatory element in the RNA genome, but the basis for protein-dependence is unclear. To address this, here we investigate structure and dynamics of the PRF signal in Theiler's murine encephalitis virus (TMEV). By combining X-ray crystallography, small angle X-ray scattering (SAXS) and single-molecule fluorescence resonance energy transfer (smFRET), we show that 2A binding switches the RNA from a stem-loop conformation into a pseudoknot, and we demonstrate that pseudoknot formation is essential for efficient PRF in vitro and in cells. Together, these findings illustrate how the cardiovirus PRF element behaves as a protein-dependent riboswitch, defining the molecular mechanism by which frameshifting is conditionally activated.

molecular biology↗

bioRxiv2024

An intra-family conserved high-order RNA structure within the M ORF is important for arterivirus subgenomic RNA accumulation and infectious virus production

Synthesis of subgenomic RNAs is a strategy commonly used by polycistronic positive sense single-stranded RNA viruses to express 3'-proximal genes. Members of the order of Nidovirales, including coronaviruses and arteriviruses, use a unique discontinuous transcription strategy to synthesize subgenomic RNAs. In this study, in silico synonymous site conservation analysis and RNA structure folding predicted the existence of intra-family conserved high-order RNA structure within the M ORF of arteriviral genomes, which was further determined to be important for the transcription/accumulation of subgenomic RNAs and production of infectious viral particles. Mutations disrupting the stability of the RNA structures significantly decreased the accumulation of multiple subgenomic RNAs. In contrast, the impact of mutagenesis on full-length genomic RNA accumulation was limited. The degree to which wild-type levels of subgenomic RNA accumulation were maintained was found to correlate with the efficiency of infectious virus production. Moreover, the thermo-stability of stems within the high-order RNA structure is also well correlated with viral replication capacity and the maintenance of subgenomic RNA accumulation. This study is the first to report an intra-Arteriviridae conserved high-order RNA structure that is located in a protein-coding region and functions as an important cis-acting element to control the accumulation/transcription of arteriviral subgenomic RNAs. This work suggests a complex regulation mechanism between genome replication and discontinuous transcription in nidoviruses. IMPORTANCEArteriviruses are a group of RNA viruses that infect different animal species. They can cause diseases associated with respiratory/reproductive syndromes, abortion, or haemorrhagic fever. Among arteriviruses, porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) are economically important veterinary pathogens. The challenge in control of arterivirus infection reflects our limited knowledge of viral biology. In this study, we conducted a comprehensive bioinformatical analysis of arteriviral genomes and discovered intra-family conserved regions in the M ORF with a high-order RNA structure. The thermo-stability of the RNA structure influences sgRNA transcription/accumulation and correlates with the level of infectious virus production. Our studies provide a new insight on arterivirus replication mechanism, which may have implications in developing disease control and prevention strategies.

molecular biology↗

bioRxiv2021

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus which causes significant economic losses to the swine industry worldwide. Here, we use ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and analyse the host response to infection. We quantified viral gene expression over a timecourse of infection, and calculated the efficiency of programmed ribosomal frameshifting (PRF) at both sites on the viral genome. At the nsp2 frameshift site (a rare example of protein-stimulated frameshifting), -2 PRF efficiency increases over time, likely facilitated by accumulation of the PRF- stimulatory viral protein (nsp1{beta}) during infection. This marks arteriviruses as the second example of temporally regulated PRF. Surprisingly, we also found PRF efficiency at the canonical ORF1ab frameshift site increases over time, in apparent contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a 125-codon ORF overlapping nsp12, which is expressed as highly as nsp12 itself at late stages of replication, and is likely translated from novel subgenomic (sg) RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV- 2, suggesting a potential conserved mechanism for temporal regulation of expression of the 3'-proximal region of ORF1b. In addition, we identified a highly translated, short upstream ORF (uORF) in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. This is the first application of RiboSeq to arterivirus-infected cells, and reveals new features which add to the complexity of gene expression programmes in this important family of nidoviruses.

pathology↗

bioRxiv2020

Structural studies of Cardiovirus 2A protein reveal the molecular basis for RNA recognition and translational control

Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein: a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to and stabilises the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we define the conformational repertoire of this element and measure changes in unfolding pathways arising from mutation and 2A binding. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, expand our understanding of the 2A-dependent gene expression switch and reveal how 2A accumulation may shut down translation during virus infection.

molecular biology↗

bioRxiv2020

Structural and molecular basis for protein-stimulated ribosomal frameshifting in Theiler's murine encephalomyelitis virus

The 2A protein of Theilers murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed -1 ribosomal frameshifting (PRF) during infection. Here we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.

molecular biology↗

bioRxiv2019

Upstream translation initiation expands the coding capacity of segmented negative-strand RNA viruses

Segmented negative-strand RNA viruses (sNSVs) include the influenza viruses, the bunyaviruses, and other major pathogens of humans, other animals and plants. The genomes of these viruses are extremely short. In response to this severe genetic constraint, sNSVs use a variety of strategies to maximise their coding potential. Because the eukaryotic hosts parasitized by sNSVs can regulate gene expression through low levels of translation initiation upstream of their canonical open reading frames (ORFs), we asked whether sNSVs could use upstream translation initiation to expand their own genetic repertoires. Consistent with this hypothesis, we showed that influenza A viruses (IAVs) and bunyaviruses were capable of upstream translation initiation. Using a combination of reporter assays and viral infections, we found that upstream translation in IAVs can initiate in two unusual ways: through non-AUG initiation in virally encoded untranslated regions, and through the appropriation of an AUG-containing leader sequence from host mRNAs through viral cap-snatching, a process we termed start-snatching. Finally, while upstream translation of cellular genes is mainly regulatory, for sNSVs it also has the potential to create novel viral gene products. If in frame with a viral ORF, this creates N-extensions of canonical viral proteins. If not, it allows the expression of cryptic overlapping ORFs, which we found were highly conserved in IAV and widely distributed in peribunyaviruses. Thus, by exploiting their hosts capacity for upstream translation initiation, sNSVs can expand still further the coding potential of their extremely compact RNA genomes.

molecular biology↗

bioRxiv2019

Comparative analysis of gene expression in virulent and attenuated strains of infectious bronchitis virus at sub-codon resolution

Infectious bronchitis virus (IBV) is a member of the genus Gammacoronavirus and the causative agent of avian infectious bronchitis. IBV has a single-stranded, positive-sense RNA genome ~27 kb in length and, like all coronaviruses, produces a set of sub-genomic messenger RNAs (sgmRNAs) synthesised via the viral polymerase. Here, we used RNA sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-CK strains at sub-codon resolution. Quantification of reads flanking the programmed ribosomal frameshifting (PRF) signal at the genomic RNA ORF1a/ORF1b junction revealed that PRF in IBV is highly efficient (33-40%), consistent with in vitro measurements. Triplet phasing of the profiling data allowed precise determination of reading frames and revealed the translation of two intergenic genes (4b and 4c on sgmRNA4), which are widely conserved across IBV isolates. RNASeq revealed two novel transcription junction sites in the attenuated Beau-CK strain, one of which would generate a sgmRNA encoding a ribosomally occupied ORF in the viral 3 untranslated region (dORF). Within IBV transcripts, the nucleocapsid (N) protein was unexpectedly found to be inefficiently translated, despite being an abundant structural component of mature IBV virions. Finally, we demonstrate that the host cell response to IBV occurs primarily at the level of transcription, with a global up-regulation of immune-related mRNA transcripts following infection, and comparatively modest changes in the translation efficiencies of host genes.\n\nIMPORTANCEIBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this pathogen, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in both pathogenic and attenuated forms of IBV, and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intra-species variation, with single nucleotide polymorphisms associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. We also identify numerous cellular genes and gene networks that are differentially expressed during virus infection, giving insights into the host cell reponse to IBV infection.

genomics↗

bioRxiv2018

Activation of the unfolded protein response and inhibition of translation initiation during coronavirus infection

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1 and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release [~]1,000-fold. Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection. Author SummarySARS-CoV-2 is the novel coronavirus responsible for the COVID-19 pandemic which has resulted in over 100 million cases since the end of 2019. Most people infected with the virus will experience mild to moderate respiratory illness and recover without any special treatment. However, older people, and those with underlying medical problems like chronic respiratory disease are more likely to develop a serious illness. So far, more than 2 million people have died of COVID-19. Unfortunately, there is no specific medication for this viral disease. In order to produce viral proteins and to replicate their genetic information, all coronaviruses use a cellular structure known as the endoplasmic reticulum or ER. However, the massive production and modification of viral proteins stresses the ER and this activates a compensatory cellular response that tries to reduce ER protein levels. This is termed the unfolded protein response or UPR. We believe that coronaviruses take advantage of the activation of the UPR to enhance their replication. The UPR is also activated in some types of cancer and neurodegenerative disorders and UPR inhibitor drugs have been developed to tackle these diseases. In this work, we have tested some of these compounds in human lung cells infected with SARS-CoV-2 and found that virus production was reduced 1000-fold in human lung cells.

microbiology↗

bioRxiv2017

The translational landscape of Zika virus during infection of mammalian and insect cells

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus recently associated with congenital diseases and neurological complications. As for all flaviviruses, the ZIKV RNA genome is expected to encode a single polyprotein with all the enzymatic activities required for viral replication. Here, we report the discovery of multiple non-canonical open reading frames (ORFs) identified by ribosome profiling. In both mammalian and insect cells infected with Asian/American and African ZIKV strains, we observed translation of previously unrecognised upstream ORFs (uORFs) in the 5' region. In the Asian/American ZIKV lineage, ribosomes translated uORF1 and uORF2 that initiated from non-AUG start codons, whereas in the African ZIKV lineage, these two uORFs were fused into a single uORF (African uORF). Using a reverse genetics system, we examined the impact on ZIKV fitness of the expression of single or dual uORFs by analysing a panel of mutant viruses. We found that expression of the African uORF, and more significantly, the Asian/American uORF1, modulated virus growth and tropism in human cortical neurons and 3D organoid tissue, indicating that these novel uORFs contribute to ZIKV neurotropism. Although ZIKV uORFs are expressed in mosquito cells, they did not have a detectable effect on transmission by the mosquito vector in vivo. Our discovery of ZIKV uORFs sheds new light on ZIKV-induced neuropathogenesis and raises the question of their existence in other neurotropic flaviviruses.

microbiology↗