Biology subjects

Routh, A. L.

Publications and source records attributed to Routh, A. L..

At least 19 recordsLinked to original sources

bioRxiv2025

Ordered release of genomic RNA during icosahedral virus disassembly

To release their genomic cargo, many icosahedral viruses undergo a series of ordered conformational changes via distinct disassembly intermediates that allow nucleic acid egress. Previous studies have focused on the rearrangement of the virus capsid protein shell during disassembly. However, it is unclear whether the packaged viral nucleic acids also undergo defined rearrangements or whether specific regions of the viral genome are released in a predetermined order. In this study, we established a Next-Generation Sequencing platform ("PT-ClickSeq") that does not require RNA extraction or fragmentation and so can natively sequence the nucleic acid exposed during virus particle disassembly without disrupting the capsid protein. We used Flock House virus (FHV) as a model system, which produces two well-defined disassembly intermediates with externalized RNA. With PT-ClickSeq, we found that FHV releases RNA genome in an ordered and conserved fashion with the 5 and 3 terminal regions of viral genomic RNAs and specific loci within the viral genome being released first during virus particle disassembly. Further, different genomic loci exhibit distinct energy barriers prior to release, suggesting a programmed exposure of the viral RNA. We also characterized viral RNA-capsid interactions using established cross-linking and NGS approaches ("vPAR-CL") and observed a disordering of RNA-capsid interactions during disassembly. Interestingly, we noticed an anti-correlation between preferentially released RNA regions and strong RNA-capsid interactions observed prior to disassembly. These findings demonstrate that rather than being a passive cargo, the encapsidated viral genome serves an important role in programmed virus disassembly. SignificanceViruses need to strike a balance between structural rigidity and flexibility, to achieve both sufficient protection and rapid release of packaged genome into host cells. During the process of genome delivery, many viruses undergo a programed disassembly process through successive morphological changes, which give rise to partially dissembled virus particles, termed disassembly intermediates. It is important to study these intermediates as "checkpoints" to understand virus disassembly dynamics. We established a next-generation sequencing method that can monitor the RNA behavior during these conformational changes. We found that different regions of RNA were released with different energy thresholds and the RNA release prioritized regions with low RNA-protein interactions. These findings shed light on the active role of the viral RNA in virus disassembly.

microbiology↗

bioRxiv2024

SARS-CoV-2 EndoU-ribonuclease regulates RNA recombination and impacts viral fitness.

Coronaviruses (CoVs) maintain large RNA genomes that frequently undergoes mutations and recombination, contributing to their evolution and emergence. In this study, we find that SARS-CoV-2 has greater RNA recombination frequency than other human CoVs. In addition, coronavirus RNA recombination primarily occurs at uridine (U)-enriched RNA sequences. Therefore, we next evaluated the role of SARS-CoV-2 NSP15, a viral endonuclease that targets uridines (EndoU), in RNA recombination and virus infection. Using a catalytically inactivated EndoU mutant (NSP15H234A), we observed attenuated viral replication in vitro and in vivo. However, the loss of EndoU activity also dysregulated inflammation resulting in similar disease in vivo despite reduced viral loads. Next-generation sequencing (NGS) demonstrated that loss of EndoU activity disrupts SARS-CoV-2 RNA recombination by reducing viral sub-genomic message but increasing recombination events that contribute to defective viral genomes (DVGs). Overall, the study demonstrates that NSP15 plays a critical role in regulating RNA recombination and SARS-CoV-2 pathogenesis.

microbiology↗

bioRxiv2023

RpoS activates Salmonella Typhi biofilms and drives persistence in a Zebrafish model

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway established in S. Typhimurium. We undertook a genome-wide Tn5 mutation screen in H58, a clinically relevant multidrug resistance strain of S. Typhi, in gallstone-mimicking conditions. We generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. An rpoS null mutant failed to colonize the gall bladder in chronic zebrafish infections. Overall, our work uncovered a novel RpoS-driven, CsgD-independent paradigm for the formation of cholesterol-attached Typhi biofilms, and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections. Our identification of the biofilm regulators in S. Typhi paves the way for the development of drugs against typhoid carriage, which will ultimately control the increased incidence of gall bladder cancer in typhoid carriers.

microbiology↗

bioRxiv2023

Loss-of-function mutation in Omicron variants reduces spike protein expression and attenuates SARS-CoV-2 infection

SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel amino acid mutations in the spike protein alone. While most studies focus on receptor binding domain changes, mutations in the C-terminus of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. In this study, we examined three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we found that the mutant increased spike processing, consistent with prior reports for H655Y and P681H individually. Next, we generated a single N679K mutant, finding reduced viral replication in vitro and less disease in vivo. Mechanistically, the N679K mutant had reduced spike protein in purified virions compared to wild-type; spike protein decreases were further exacerbated in infected cell lysates. Importantly, exogenous spike expression also revealed that N679K reduced overall spike protein yield independent of infection. Although a loss-of-function mutation, transmission competition demonstrated that N679K had a replication advantage in the upper airway over wild-type SARS-CoV-2 in hamsters, potentially impacting transmissibility. Together, the data show that N679K reduces overall spike protein levels during Omicron infection, which has important implications for infection, immunity, and transmission.

microbiology↗

bioRxiv2022

SARS-CoV-2 Uses Nonstructural Protein 16 to Evade Restriction by IFIT1 and IFIT3

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2-O methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2-O MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2-O methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, a methyltransferase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a possible target for future antiviral therapies. ImportanceSimilar to other coronaviruses, disruption of SARS-CoV-2 NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1, but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2-O methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

microbiology↗

bioRxiv2022

Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability

Diverse RNA-RNA interactions occur throughout the lifecycle of RNA viruses, including genome dimerization or multimerization through non-covalent association of two or more genomic segments. Both homo-dimerization of retrovirus genomic RNA and hetero-multimerization of genomic segments of multipartite viruses are recognized as important factors that govern genome packaging. The heterodimer of the bipartite alphanodaviruses represents a unique case as its formation is conserved among different virus species but is only observed within the context of fully assembled virus particles. In spite of this, the RNA sequences involved in heterodimerization are unknown. It also remains unclear whether the formation of this heterodimer might impact any stage of the virus lifecycle. In this study, we used Flock House virus (FHV) as a model system to uncover the nucleotide composition of the heterodimer and dissected the impact of heterodimer formation upon numerous stages of the virus lifecycle. We developed a novel Next-Generation Sequencing (NGS) approach called "XL-ClickSeq" to probe candidate heterodimer sequences. We found that the heterodimer is formed via intermolecular base-pairing and its formation is retained in defective virus particles. One heterodimer site was identified by thermodynamic prediction that, upon mutagenic disruption, exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNA. Furthermore, we demonstrate that disruption of this RNA secondary structure directly impacts the thermostability of mature virus particles. These results demonstrated that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus can have an important impact on virus particle integrity and stability and thus its transmission to a new host.

molecular biology↗

bioRxiv2022

ViReMaShiny: An Interactive Application for Analysis of Viral Recombination Data

Recombination is an essential driver of virus evolution and adaption, giving rise to new chimeric viruses, structural variants, sub-genomic RNAs, and Defective-RNAs. Next-Generation Sequencing of virus samples, either from experimental or clinical settings, has revealed a complex distribution of recombination events that contributes to the intrahost diversity. We and others have previously developed alignment tools to discover and map these diverse recombination events in NGS data. However, there is no standard for data visualization to contextualize events of interest and downstream analysis often requires bespoke coding. To address this, we present ViReMaShiny, a web-based application built using the R Shiny framework to allow interactive exploration and point-and-click visualization of viral recombination data provided in BED format generated by computational pipelines such as ViReMa (Viral-Recombination-Mapper).

bioinformatics↗

bioRxiv2022

ViReMa: A Virus Recombination Mapper of Next-Generation Sequencing data characterizes diverse recombinant viral nucleic acids

Genetic recombination is a tremendous source of intra-host diversity in viruses and is critical for their ability to rapidly adapt to new environments or fitness challenges. While viruses are routinely characterized using high-throughput sequencing techniques, characterizing the genetic products of recombination in next-generation sequencing data remains a challenge. Viral recombination events can be highly diverse and variable in nature, including simple duplications and deletions, or more complex events such as copy/snap-back recombination, inter-virus or inter-segment recombination and insertions of host nucleic acids. Due to the variable mechanisms driving virus recombination and the different selection pressures acting on the progeny, recombination junctions rarely adhere to simple canonical sites or sequences. Furthermore, numerous different events may be present simultaneously in a viral population, yielding a complex mutational landscape. We have previously developed an algorithm called ViReMa (Virus Recombination Mapper) that bootstraps the bowtie short-read aligner to capture and annotate a wide-range of recombinant species found within virus populations. Here, we have updated ViReMa to provide an error-density function designed to accurately detect recombination events in the longer reads now routinely generated by the Illumina platforms and provide output reports for multiple types of recombinant species using standardized formats. We demonstrate the utility and flexibility of ViReMa in different settings to report deletion events in simulated data from Flock House virus, copy-back RNA species in Sendai viruses, short duplication events in HIV, and virus to host recombination in an archaeal DNA virus.

bioinformatics↗

bioRxiv2022

Selective Ablation of 3' RNA ends and Processive RTs Facilitate Direct cDNA Sequencing of Full-length Host cell and Viral Transcripts

Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read-lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3 RNA ends (termed CASPR) which decreases ribosomal RNA reads and enriches for poly-adenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.

genomics↗

bioRxiv2021

QTQTN motif upstream of the furin-cleavage site plays key role in SARS-CoV-2 infection and pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing 1,2. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates 3. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif ({Delta}QTQTN). Here we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS, and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated4, and disruption its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site - the FCS, loop length, and glycosylation - are required for efficient SARS-CoV-2 replication and pathogenesis.

microbiology↗

bioRxiv2021

Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis.

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral RG motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2s continued adaptation to human infection. Author SummarySince its emergence, SARS-CoV-2 has continued to adapt for human infection resulting in the emergence of variants with unique genetic profiles. Most studies of genetic variation have focused on spike, the target of currently available vaccines, leaving the importance of variation elsewhere understudied. Here, we characterize a highly variable motif at residues 203-205 in nucleocapsid. Recreating the prominent nucleocapsid R203K+G204R mutation in an early pandemic background, we show that this mutation is alone sufficient to enhance SARS-CoV-2 replication and pathogenesis. We also link augmentation of SARS-CoV-2 infection by the R203K+G204R mutation to its modulation of nucleocapsid phosphorylation. Finally, we characterize an analogous alanine double substitution at positions 203-204. This mutant was found to mimic R203K+G204R, suggesting augmentation of infection occurs by disrupting the ancestral sequence. Together, our findings illustrate that mutations outside of spike are key components of SARS-CoV-2s adaptation to human infection.

microbiology↗

bioRxiv2021

Co-variation of viral recombination with single nucleotide variants during virus evolution revealed by CoVaMa

Adaptation of viruses to their environments occurs through the acquisition of both novel Single-Nucleotide Variants (SNV) and recombination events including insertions, deletions, and duplications. The co-occurrence of SNVs in individual viral genomes during their evolution has been well-described. However, unlike covariation of SNVs, studying the correlation between recombination events with each other or with SNVs has been hampered by their inherent genetic complexity and a lack of bioinformatic tools. Here, we expanded our previously reported CoVaMa pipeline (v0.1) to measure linkage disequilibrium between recombination events and SNVs within both short-read and long-read sequencing datasets. We demonstrate this approach using long-read nanopore sequencing data acquired from Flock House virus (FHV) serially passaged in vitro. We found SNVs that were either correlated or anti-correlated with large genomic deletions generated by nonhomologous recombination that give rise to Defective-RNAs. We also analyzed NGS data from longitudinal HIV samples derived from a patient undergoing antiretroviral therapy who proceeded to virological failure. We found correlations between insertions in the p6Gag and mutations in Gag cleavage sites. This report confirms previous findings and provides insights on novel associations between SNVs and specific recombination events within the viral genome and their role in viral evolution.

genetics↗

bioRxiv2021

Mouse Adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge

The emergence of SARS-CoV-2 has resulted in a worldwide pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of COVID-19 disease have been hampered by the lack of robust mouse models. To overcome this barrier, we utilized a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARSCoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse-adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Utilizing this model, we demonstrate that SARS-CoV-2 infected mice are protected from lethal challenge with the original SARS-CoV, suggesting immunity from heterologous CoV strains. Together, the results highlight the utility of this mouse model for further study of SARS-CoV-2 infection and disease.

microbiology↗

bioRxiv2021

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called Tiled-ClickSeq, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

molecular biology↗

bioRxiv2021

MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution

Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolution given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of long-range genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure, and enable the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

genomics↗

bioRxiv2020

Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis

SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site ({Delta}PRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the {Delta}PRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the {Delta}PRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays. ImportanceAs COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis. Article SummaryA deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.

microbiology↗

bioRxiv2020

RBFOX2 Is Critical For Maintaining Alternative Polyadenylation And Mitochondrial Health In Myoblasts

RBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression/function in muscle cells, contributing to disease pathology. Here, we employed both 3-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. We found that RBFOX2-mediated APA modulates both mRNA levels and isoform expression of a collection of genes including contractile and mitochondrial genes. We identified the key muscle-specific contractile gene, Tropomyosin 1 and essential mitochondrial gene, Slc25a4 as APA targets of RBFOX2. Unexpectedly, depletion of RBFOX2 adversely affected mitochondrial health in myoblasts that is in part mediated by disrupted APA of mitochondrial gene Slc25a4. Mechanistically, we found that RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site enforcing the use of the proximal polyadenylation site. In sum, our results unveiled a new role for RBFOX2 in fine tuning expression levels of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases.

molecular biology↗

bioRxiv2020

The coronavirus proofreading exoribonuclease mediates extensive viral recombination

Coronaviruses (CoVs) emerge as zoonoses and cause severe disease in humans, demonstrated by the SARS-CoV-2 (COVID-19) pandemic. RNA recombination is required during normal CoV replication for subgenomic mRNA (sgmRNA) synthesis and generates defective viral genomes (DVGs) of unknown function. However, the determinants and patterns of CoV recombination are unknown. Here, we show that divergent {beta}-CoVs SARS-CoV-2, MERS-CoV, and murine hepatitis virus (MHV) perform extensive RNA recombination in culture, generating similar patterns of recombination junctions and diverse populations of DVGs and sgmRNAs. We demonstrate that the CoV proofreading nonstructural protein (nsp14) 3-to-5 exoribonuclease (nsp14-ExoN) is required for normal CoV recombination and that its genetic inactivation causes significantly decreased frequency and altered patterns of recombination in both infected cells and released virions. Thus, nsp14-ExoN is a key determinant of both high fidelity CoV replication and recombination, and thereby represents a highly-conserved and vulnerable target for virus inhibition and attenuation.

microbiology↗