Biology subjects

Gaunt, E. R.

Publications and source records attributed to Gaunt, E. R..

12 recordsLinked to original sources

bioRxiv2026

The biochemical composition of coronavirus Nucleocapsid proteins drives the bicistronic properties of their sub-genomic mRNAs

The nucleocapsid (N) gene of coronaviruses (CoVs) encodes a structural RNA-binding protein required for viral assembly. In SARS- and MERS-CoV viruses, reading frame 2 of N contains an overlapping open reading frame (ORF) encoding an innate immune antagonist accessory protein. However, the extent of this dual coding capacity across the CoV family, and the genetic features driving overlapping ORF formation, remain unexamined. Here, we developed an in-silico tool (ORFan) to screen structural gene sub-genomic mRNA sequences from 152 species of coronavirus for the presence of overlapping accessory gene candidates. We found that frame 2 of the N gene in Alpha-, Beta- and Delta-CoVs frequently contains a potential accessory ORF (AcORF), conserved at the subgenus level, unlike the spike, envelope and membrane genes. We also show that CoV nucleotide biases in conjunction with codon biases integral to N protein function drive the relatively high abundances of start codons in frame two and stop codons in frame three, explaining the tendency towards frame 2 AcORFs. We further suggest that viral nucleotide biases and the codon composition required for RNA-binding function facilitate overlapping ORF formation in other virus families

microbiology↗

bioRxiv2026

Investigating the evolutionary plasticity of influenza virus splicing using influenza A virus segment 7 as a model system

Influenza viruses A, C and D (IAV, ICV, IDV respectively) utilise mRNA splicing to produce two proteins from their matrix gene segment; the matrix (M1) protein and an ion channel (M2). In IAV, the M1 and M2 open reading frames (ORFs) partially overlap and splice donor (SD) and splice acceptor (SA) sites in the M1 gene are used to produce an M2 polypeptide with an N-terminal sequence shared with M1. In ICV and IDV, the M1 and M2 ORFs are concatenated and splice sites downstream of the M1 coding region are used to produce a matrix protein from the spliced mRNA. To probe how these different splicing strategies might have evolved, we used reverse genetics to test the evolutionary flexibility of IAV SD and SA sites. We found that relatively small numbers of mutations were sufficient to activate a variety of alternative SD and SA sites in a mini-replicon system, several of which led to the production of ion channel isoforms with variant ectodomains, as well as one that produced an M1 variant. Furthermore, several of the viruses with altered splicing patterns were viable, including five with both SD and SA sites changed. Three of these 'rewired' viruses had only modest fitness penalties, despite up to 3 nonsynonymous changes in M1 and a 34 amino acid insertion in M2. These results suggest that IAV segment 7 splicing strategies are evolutionarily flexible and have implications for how IAV, ICV and IDV matrix segments might have diversified from a common ancestor.

microbiology↗

bioRxiv2026

Splice-Aware Optimization Prevents Pervasive Missplicing of Natural and Synthetic cDNAs

Heterologous gene expression is widely used across biology and medicine, and often relies on codon optimization to increase protein yields. Here we uncover missplicing as a common and largely unrecognized failure mode of heterologous expression. Using systematically designed libraries comprising over 5,000 synthetic reporter genes and natural human cDNAs, we find that the majority of gene variants expressed in a human cell line are at least partially spliced, and in many variants the spliced isoform dominates, reducing protein output or ablating expression entirely. By analysing sequence determinants of expression across multiple human cell lines, we uncover a hierarchical architecture of regulatory control, where GC content establishes baseline mRNA levels, local sequence features influence splicing, and tissue-specific codon adaptation to tRNA pools fine-tunes translation efficiency. These findings enable us to develop predictive models of expression and splicing, benchmark current optimization strategies, and design a splice-aware optimization algorithm that substantially improves transgene performance.

synthetic biology↗

bioRxiv2025

Identification of cis-Acting Elements that Regulate Influenza A Virus Segment 7 Differential mRNA Splicing

Influenza A virus segment 7 encodes the M1 matrix protein and M2 ion channel from an unspliced primary transcript mRNA1 and spliced mRNA2, respectively. Both proteins are essential for efficient virus replication, but the intrinsic sequences that determine segment 7 splicing balance are not fully defined. Here, we systematically investigated how conserved cis-acting splicing elements regulate 7 splicing. We found that while the splice acceptor sequence is optimal, both potential upstream polypyrimidine tracts regulate splicing, the 5 region acting positively and the 3 region negatively to maintain M1 expression. We could not unambiguously identify the mRNA2 branch point, but two closely spaced motifs can act redundantly. Saturation mutagenesis of the mRNA2 splice donor sequence showed that it is nearly optimal but that nucleotides 51 and 55 modestly downregulate splicing efficiency. Most introduced mutations that change splicing balance are nonsynonymous in M1. Nevertheless, mutations in the polypyrimidine tract and branch points that significantly perturbed M1:M2 balance resulted in viable viruses. In contrast, the splice donor sequence was intolerant of mutation. We conclude that M1:M2 balance is maintained via suboptimal splice donor and polypyrimidine tract splicing signals, with the latter allowing more evolutionary flexibility to vary the ratio and/or M1 sequence.

microbiology↗

bioRxiv2025

The cow udder is a potential mixing vessel for influenza A viruses

The incursion of high pathogenicity avian influenza A virus (IAV) into US dairy cows is unprecedented in the era of molecular diagnosis and pathogen sequencing. This raises questions over the likelihood of further outbreaks and whether dairy cattle could be a "mixing vessel" for novel strains of IAV. Using a panel of BSL2-safe reassortant viruses representing clade 2.3.4.4b H5 epizootic lineages circulating since 2020, we found that a cow B3.13 isolate displayed enhanced replication in cow mammary gland cells, along with increased viral polymerase activity and stronger interferon antagonism in cow cells compared to an earlier EA-2020-C genotype virus. However, multiple avian and mammalian IAV strains, including other clade 2.3.4.4b high pathogenicity genotypes, were replication competent in bovine cells, particularly those of the mammary gland, suggesting that there is a diverse circulating IAV pool with the potential to infect cows. Moreover, we show that cow mammary cells co-express -2,3 and -2,6 - linked sialic acids, and are susceptible to co-infection with human and avian IAVs. We conclude that the US cow influenza outbreak does not simply reflect a unique adaptation of the B3.13 genotype virus; rather, the bovine udder represents a permissive niche for IAV and a plausible site for reassortment, underscoring its potential role in generating novel influenza viruses with pandemic risk.

microbiology↗

bioRxiv2025

Synergy between HA cleavage site sequence and NA-mediated plasminogen recruitment as a virulence mechanism for low pathogenic avian influenza

An outbreak of H3N1 low pathogenic avian influenza virus (LPAIV) in Belgium in 2019 caused unexpected levels of mortality and morbidity in poultry. These viruses possess an NA polymorphism associated with plasminogen binding, as well as an unusual sequence around the HA cleavage site; accordingly, HA cleavage mediated by NA-driven plasminogen recruitment has been proposed to underly their systemic spread and pathogenicity. To test this, we established a reverse genetics system for A/chicken/Belgium/460/2019 and created single mutations in HA (K345R), and NA (S122N) that restored the viruses to normal consensus, as well as an HA/NA double mutant. Confirming previous work, trypsin-independent spread and HA cleavage of wild type Ck/Belgium was observed in the presence of fetal bovine serum containing plasminogen in vitro. Dose-dependent HA cleavage and trypsin-independent spread was also observed in the presence of purified chicken plasminogen. Compared to wild type virus, both HA cleavage and virus spread in vitro were reduced by the HA K345R mutation and further blocked by NA mutation S122N. Plasminogen-mediated HA cleavage was seen in a variety of avian cell lines and chicken organoids, excluding cell type-dependent effects. Furthermore, in ovo tests showed that mutant viruses unable to recruit plasminogen were less able to replicate systemically in chicken embryos. Bioinformatics analyses revealed other viruses which could potentially recruit plasminogen, including two independent outbreaks of H6N1 viruses, one of which we confirmed PLG-driven spread in vitro. We conclude that PLG-recruitment by NA is a general virulence mechanism of N1 LPAIVs Author summaryAvian influenza viruses (AIV) are divided into two broad categories - high or low pathogenicity - based on the sequence of their haemagglutinin (HA) and their lethality in chickens. The majority of AIV strains circulating in the wild are low pathogenicity both in waterfowl and when they spill over into domestic poultry. However, some low pathogenicity strains can cause severe disease in poultry despite not being classified as H5 or H7 high pathogenic AIVs with an HA polybasic protease cleavage site. A severe 2019 outbreak of an H3N1 strain has been suggested to result from the neuraminidase (NA) of the virus recruiting cellular plasminogen to proteolytically activate HA. Here, we tested this hypothesis by using reverse genetics to mutate the virus in a way predicted to block this. We found that indeed, the sequence of the NA at position 122 is the primary determinant of plasminogen-driven HA cleavage but that the unusual sequence at the HA cleavage site of the outbreak virus also contributes to pathogenicity. Furthermore, we show that N1 NA sequence can be used to identify other unexpectedly virulent strains of AIV. This work therefore adds to our ability to risk assess AIV strains from sequence-based surveillance.

microbiology↗

bioRxiv2023

Understanding off-target growth defects introduced to influenza A virus by synonymous recoding

CpG dinucleotides are under-represented in the genomes of most RNA viruses. Synonymously increasing CpG content of a range of RNA viruses reliably causes replication defects due to the recognition of CpG motifs in RNA by cellular Zinc-finger Antiviral Protein (ZAP). Prior to the discovery of ZAP as a CpG sensor, we described an engineered influenza A virus (IAV) enriched for CpGs in segment 5 that displays the expected replication defects. However, we report here that this CpG-high ( CpGH) mutant is not attenuated by ZAP. To understand this, we sought to uncover the alternative attenuation mechanism(s). IAV segment 5 encodes NP, a component of the viral RNA replication complex. Unexpectedly, while CpG enrichment resulted in depleted segment 5 transcript and NP protein abundance, this did not impair viral polymerase activity. A pair of nucleotide changes, introduced as compensatory changes to maintain base frequencies, were instead found to be responsible for the replication defect. These mutations resulted in the encoding of a stretch of eight consecutive adenosines (8A), a phenomenon not seen in natural IAV isolates. Sequencing experiments revealed evidence of viral polymerase slippage at this site, resulting in the production of aberrant peptides and type I interferon induction. When the nucleotides in either of these two positions were restored to wildtype sequence, no viral attenuation was seen, despite the 86 extra CpGs encoded by this virus. Conversely, when these two adenosines were introduced into wildtype virus (thereby introducing the 8A tract), viral attenuation, polymerase slippage, aberrant peptide production and type I interferon induction were apparent. That a single nucleotide change can offset the growth defects in a virus designed to have a formidable barrier to wild-type reversion highlights the importance of understanding the processes underlying viral attenuation. The lessons from this study will inform improved recoding designs in the future.

microbiology↗

bioRxiv2022

Synthetically engineered IgG1 antibody Fc fragments presenting influenza A virus receptor sialic acid inhibit viral haemagglutination activity, but enhance virus replication in cultured A549 cells

Many clinically important viruses, including influenza A, SARS-CoV-1, adenoviruses, and DNA tumour viruses such as Kaposis sarcoma herpesvirus use multivalent binding to sialic acid (SA) to infect cells, or to modulate immune responses through interactions with sialylated attachment factors that facilitate virus infectivity and/or host survival. Molecular scaffolds rich in SA that bind virions with high avidity may therefore be useful as anti-infective medicines. We generated a panel of 12 of these molecules using fragment-crystallisable scaffolds in CHO-S cells that are rich in SA. The viral surface protein of influenza A virus (IAV), haemagglutinin, binds SA for cell entry, and so we tested the activity of these compounds against this virus. Two of the sialylated Fc-molecules reduced IAV haemagglutination activity by up to 64-fold. However, the same molecules enhanced virus infectivity of A549 cultured cells. To explain the increased viral titres, we postulated that sialylated Fcs may be anti-inflammatory. However, sialylated Fc multimers were instead pro-inflammatory; they induced chemokine/cytokine responses from differentiated human THP-1 derived macrophages, including raised IL-8 and MIP-1/{beta}, that mimicked responses driven by universal type I interferon. Steric targeting of SA to block virus entry may therefore have unexpected effects in target cells that currently preclude their use for medical intervention.

immunology↗

bioRxiv2022

CpG enrichment as a vaccine strategy for influenza A virus

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP- sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines. AUTHOR SUMMARYCpG dinucleotides are under-represented in vertebrate genomes, wherein cytosines in the CpG conformation are methylated to regulate transcription. Methylated cytosines are prone to deamination, resulting in TpG dinucleotides replacing CpGs. The resultant CpG suppression has provided a route by which vertebrate cells can recognise RNA from invading pathogens, using cellular Zinc-finger Antiviral Protein (ZAP) as a CpG sensor. Vertebrate-infecting RNA viruses also genomically suppress CpGs, and it is believed that this is an evolved trait to evade detection by ZAP. Here, we engineered an influenza A virus (IAV) with elevated CpG content and characterised how this impacts viral replication. CpG addition resulted in viral attenuation, mediated by ZAP activity. CpG suppression is conserved in dog and chicken genomes (relevant for live attenuated IAV vaccine propagation), and it is logical to predict that ZAP-mediated CpG sensing would also be conserved in these species. However, when we propagated ZAP-sensitive IAV in cognate culture systems, we saw no replication defect. This unexpected result raises questions about why viruses infecting these species suppress CpG in their genomes, and importantly delivers a new, tractable approach to augment rational live attenuated IAV vaccine design.

microbiology↗

bioRxiv2021

TRIM25 inhibits Influenza A infection by destabilising its mRNA and is redundant for the RIG-I pathway.

The E3 ubiquitin ligase TRIM25 is a key factor in the innate immune response to RNA viruses. TRIM25 has been shown to play a role in the retinoic-acid-inducible gene-1 (RIG-I) pathway, which triggers expression of type 1 interferons upon viral infection. We and others have recently shown that TRIM25 is an RNA-binding protein, however not much is known about the RNA-binding roles of TRIM25 in the innate immune response to RNA viruses. Here, we demonstrate that influenza A virus (IAV A/PR/8/34_NS1(R38K41A)) infection is inhibited by TRIM25. Surprisingly, host RNA-binding deficient mutant TRIM25{Delta}RBD and TRIM25{Delta}RING, which lack E3 ubiquitin ligase activity rescued IAV inhibition in TRIM25 knock-out cells. Furthermore, we show that in human cultured cells activation of the RIG-I/interferon type 1 pathway mediated by an IAV-derived 5-triphosphate RNA does not require TRIM25 activity. Additionally, knocking out TRIM25 does not affect the activity of the IAV polymerase. We present new evidence that TRIM25 restricts IAV by directly binding to and destabilising its mRNAs. Finally, we show that direct tethering of TRIM25 to RNA is sufficient to downregulate the targeted RNA. In summary, our results uncover a novel mechanism that TRIM25 uses to inhibit IAV infection and regulate RNA metabolism.

molecular biology↗

bioRxiv2020

STING Nuclear Partners Contribute to Innate Immune Signalling Responses

STING and cGAS initiate innate immune responses (IIR) by recognizing cytoplasmic pathogen dsDNA and activating signaling cascades from the ER; however, another less investigated pool of STING resides in the nuclear envelope. We find that STING in the inner nuclear membrane increases mobility and changes localization upon IIR activation both from dsDNA and poly(I:C) stimuli. We next identified nuclear partners of STING from isolated nuclear envelopes. These include several known nuclear membrane proteins, bromodomain and epigenetic enzymes, and RNA- or DNA-binding proteins. Strikingly, 17 of these DNA and RNA-binding STING partners are known to bind direct partners of the IRF3/7 transcription factors that are central drivers of IIR. We find that several of these STING partners --SYNCRIP, Men1, Ddx5, snRNP70, RPS27a, Aatf-- can contribute to IIR activation and SYNCRIP can moreover protect against influenza A virus infection. These data suggest that the many roles identified for STING likely reflect its interactions with multiple RNA and DNA-binding proteins that also function in IIR.

immunology↗

bioRxiv2020

Intra-genome variability in the dinucleotide composition of SARS-CoV-2

CpG dinucleotides are under-represented in the genomes of single stranded RNA viruses, and coronaviruses, including SARS-CoV-2, are no exception to this. Artificial modification of CpG frequency is a valid approach for live attenuated vaccine development, and if this is to be applied to SARS-CoV-2, we must first understand the role CpG motifs play in regulating SARS-CoV-2 replication. Accordingly, the CpG composition of the newly emerged SARS-CoV-2 genome was characterised in the context of other coronaviruses. CpG suppression amongst coronaviruses does not significantly differ according to genera of virus, but does vary according to host species and primary replication site (a proxy for tissue tropism), supporting the hypothesis that viral CpG content may influence cross-species transmission. Although SARS-CoV-2 exhibits overall strong CpG suppression, this varies considerably across the genome, and the Envelope (E) open reading frame (ORF) and ORF10 demonstrate an absence of CpG suppression. While ORF10 is only present in the genomes of a subset of coronaviruses, E is essential for virus replication. Across the Coronaviridae, E genes display remarkably high variation in CpG composition, with those of SARS and SARS-CoV-2 having much higher CpG content than other coronaviruses isolated from humans. Phylogeny indicates that this is an ancestrally-derived trait reflecting their origin in bats, rather than something selected for after zoonotic transfer. Conservation of CpG motifs in these regions suggests that they have a functionality which over-rides the need to suppress CpG; an observation relevant to future strategies towards a rationally attenuated SARS-CoV-2 vaccine.

evolutionary biology↗