Biology subjects

Mehle, A.

Publications and source records attributed to Mehle, A..

14 recordsLinked to original sources

bioRxiv2025

RtmR is a membrane-embedded RRM-family RNA-binding protein that regulates biofilm formation

The animal symbiont Vibrio fischeri has served as a model organism for molecular processes underlying bacterial group behaviors, including quorum sensing and biofilm development. Here, using a genetic approach to identify negative regulators of biofilm formation in V. fischeri, we identified a membrane-bound RNA-binding protein, RtmR (VF_2432), that acts as an inhibitor of the symbiosis polysaccharide (SYP) biofilm. Membrane localization of the protein seems to be required for protein stability or expression, as truncation of the transmembrane helices led to an inability to detect the protein. The conserved RNP1 and RNP2 motifs in RtmRs cytoplasmic RNA recognition motif (RRM) domain are required for function, and we demonstrate binding to RNA substrates. Identification of RtmR RNA ligands was conducted with a iiCLIP-seq approach that revealed a large interactome. One transcript identified was that of the biofilm regulatory histidine kinase RscS. We found that RtmR biofilm inhibition depends on RscS activity and that RtmR negatively regulates levels of RscS. Overall, this work characterizes a novel type of bacterial RNA-binding protein. IMPORTANCEBacterial RNA-binding proteins (RBPs) perform key functions to regulate stress responses and development. Bacterial RBPs including the RNA chaperones Hfq and ProQ, the global regulator CsrA, and the cold shock proteins (Csps) have been extensively studied, although additional classes of RBPs have been predicted by bioinformatic methods including those carrying an RRM domain. This work expands on recent studies of RRM domain proteins in bacteria to characterize a membrane-bound RRM protein that regulates bacterial biofilm development. Given our rapidly-expanding knowledge regarding the role for RNA-binding proteins in bacterial molecular biology, this work contributes a new class of membrane-bound regulators with homologs in human pathogens and marine symbionts.

microbiology↗

bioRxiv2025

Influenza virus antagonizes self sensing by RIG-I to enhance viral replication

Innate immune sensors must finely distinguish pathogens from the host to mount a response only during infection. RIG-I is cytoplasmic sensor that surveils for foreign RNAs. When activated, RIG-I triggers a broad antiviral response that is a major regulator of RNA virus infection. Here were show that RIG-I not only bound viral RNAs, but was activated by host RNAs to amplify the antiviral state. These were primarily non-coding RNAs transcribed by RNA polymerase III. They were benign under normal conditions but became immunogenic during influenza virus infection where they signaled via RIG-I to suppress viral replication. This same class of RNAs was bound by influenza virus nucleoprotein (NP), which normally functions to encapsidate the viral genome. NP interacted with RIG-I and cytoplasmic NP antagonized sensing of self RNAs to counter innate immune responses. Overall, these results demonstrate that self sensing is strategically deployed by the cell to amplify the antiviral response and reveal a newly identified viral countermeasure that disrupts RIG-I activation by host RNAs.

microbiology↗

bioRxiv2025

IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

Host cells produce a vast network of antiviral factors in response to viral infection. The interferon-induced proteins with tetratricopeptide repeats (IFITs) are important effectors of a broad-spectrum antiviral response. In contrast to their canonical roles, we previously identified IFIT2 and IFIT3 as pro-viral host factors during influenza A virus (IAV) infection. During IAV infection, IFIT2 binds and enhances translation of AU-rich cellular mRNAs, including many IFN-simulated gene products, establishing a model for its broad antiviral activity. But, IFIT2 also bound viral mRNAs and enhanced their translation resulting in increased viral replication. The ability of IFIT3 to bind RNA and whether this is important for its function was not known. Here we validate direct interactions between IFIT3 and RNA using electromobility shift assays (EMSAs). RNA-binding site identification (RBS-ID) experiments then identified an RNA-binding surface composed of residues conserved in IFIT3 orthologs and IFIT2 paralogs. Mutation of the RNA-binding site reduced the ability IFIT3 to promote IAV gene expression and translation efficiency when compared to wild type IFIT3. The functional units of IFIT2 and IFIT3 are homo- and heterodimers, however the RNA-binding surfaces are located near the dimerization interface. Using co-immunoprecipitation, we showed that mutations to these sites do not affect dimerization. Together, these data establish the link between IFIT3 RNA-binding and its ability to modulate translation of host and viral mRNAs during IAV infection. ImportanceInfluenza A viruses (IAV) cause considerable morbidity and mortality through sporadic pandemics as well as annual epidemics. Zoonotic IAV strains pose an additional risk of spillover into a naive human population where prior immunity can have minimal effect. In this case, the first line of defense in the host is the innate immune response. Interferon stimulated genes (ISGs) produce a suite of proteins that are front-line effectors of innate immune responses. While ISGs are typically considered antiviral, new work has revealed an emerging trend where viruses co-opt ISGs for pro-viral function. Here, we determine how the ISG IFIT3 is used by IAV as a pro-viral factor, advancing our understanding of IFIT3 function generally as well as specifically in the context of IAV infection.

microbiology↗

bioRxiv2024

Global remodeling of ADP-ribosylation by PARP1 suppresses influenza A virus infection

ADP-ribosylation is a highly dynamic and fully reversible post-translational modification performed by poly(ADP-ribose) polymerases (PARPs) that modulates protein function, abundance, localization and turnover. Here we show that influenza A virus infection causes a rapid and dramatic upregulation of global ADP-ribosylation that inhibits viral replication. Mass spectrometry defined for the first time the global ADP-ribosylome during infection, creating an infection-specific profile with almost 4,300 modification sites on [~]1,080 host proteins, as well as over 100 modification sites on viral proteins. Our data indicate that the global increase likely reflects a change in the form of ADP-ribosylation rather than modification of new targets. Functional assays demonstrated that modification of the viral replication machinery antagonizes its activity and further revealed that the anti-viral activity of PARPs and ADP-ribosylation is counteracted by the influenza A virus protein NS1, assigning a new activity to the primary viral antagonist of innate immunity. We identified PARP1 as the enzyme producing the majority of poly(ADP-ribose) present during infection. Influenza A virus replicated faster in cells lacking PARP1, linking PARP1 and ADP-ribosylation to the anti-viral phenotype. Together, these data establish ADP-ribosylation as an anti-viral innate immune-like response to viral infection antagonized by a previously unknown activity of NS1.

microbiology↗

bioRxiv2024

A single ancestral ANP32 locus in ticks creates multiple protein variants that all support the Thogoto virus polymerase

Viral polymerases rely on cellular cofactors to support efficient transcription of viral genes and replication of the viral genome. The RNA-dependent RNA polymerase of influenza virus, an orthomyxovirus, requires cellular ANP32A or ANP32B proteins for genome replication. However, little is known about whether ANP32 proteins are required by other orthomyxoviruses like the tick-borne thogotoviruses. Recent structural studies coupled with functional assays suggested that the Thogoto virus polymerase uses both ANP32A and ANP32B from brown dog ticks. We clarify here that this tick vector encodes a single ANP32 locus corresponding to ANP32A. This single gene produces multiple protein variants through alternative splicing and start-site selection, all of which enhance Thogoto virus polymerase. Thogoto virus polymerase activity is also enhanced by human and chicken ANP32 proteins. Thus, ANP32A is a deeply conserved pro-viral cofactor and Thogoto virus shows remarkable plasticity utilizing ANP32 homologues separated by almost 1 billion years of evolution.

microbiology↗

bioRxiv2024

Multiple Unfolded Protein Response pathways cooperate to link cytosolic dsDNA release to Stimulator of Interferon Gene (STING) activation

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-{beta} expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-{beta} induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-{beta}, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-{beta} mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-{beta} via mitochondrial dsDNA release.

immunology↗

bioRxiv2023

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes as they. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

microbiology↗

bioRxiv2023

Pathogen-driven CRISPR screens identify TREX1 as a regulator of DNA self-sensing during influenza virus infection

Intracellular pathogens interact with host factors, exploiting those that enhance replication while countering those that suppress it. Genetic screens have begun to define the host:pathogen interface and establish a mechanistic basis for host-directed therapies. Yet, limitations of current approaches leave large regions of this interface unexplored. To uncover host factors with pro-pathogen functions, we developed a novel fitness-based screen that queries factors important during the middle-to-late stages of infection. This was achieved by engineering influenza virus to direct the screen by programing dCas9 to modulate host gene expression. A genome-wide screen identified the cytoplasmic DNA exonuclease TREX1 as a potent pro-viral factor. TREX1 normally degrades cytoplasmic DNA to prevent inappropriate innate immune activation by self DNA. Our mechanistic studies revealed that this same process functions during influenza virus infection to enhance replication. Infection triggered release of mitochondrial DNA into the cytoplasm, activating antiviral signaling via cGAS and STING. TREX1 metabolized the mitochondrial DNA preventing its sensing. Collectively, these data show that self-DNA is deployed to amplify host innate sensing during RNA virus infection, a process tempered by TREX1. Moreover, they demonstrate the power and generality of pathogen driven fitness-based screens to pinpoint key host regulators of intracellular pathogens.

microbiology↗

bioRxiv2021

Influenza A virus undergoes compartmentalized replication in vivo dominated by stochastic bottlenecks

Transmission of influenza A viruses (IAV) between hosts is subject to numerous physical and biological barriers that impose genetic bottlenecks, constraining viral diversity and adaptation. The presence of bottlenecks within individual hosts and their potential impacts on evolutionary pathways taken during infection and subsequent transmission are poorly understood. To address this knowledge gap, we created highly diverse IAV libraries bearing molecular barcodes on two independent gene segments, enabling high-resolution tracking and quantification of unique virus lineages within hosts. Here we show that IAV infection in lungs is characterized by multiple within-host bottlenecks that result in "islands" of infection in lung lobes, each with genetically distinct populations. We performed site-specific inoculation of barcoded IAV in the upper respiratory tract of ferrets and tracked viral diversity as infection spread to the trachea and lungs. We observed compartmentalized replication of discrete barcoded populations within the lobes of the lung. Bottlenecks stochastically sampled individual viruses from the upper respiratory tract or the trachea that became the dominant genotype in a particular lobe. These populations are shaped strongly by founder effects, with no evidence for positive selection. The segregated sites of replication highlight the jackpot-style events that contribute to within-host influenza virus evolution and may account for low rates of intrahost adaptation.

microbiology↗

bioRxiv2020

Alternative splicing liberates a cryptic cytoplasmic isoform of mitochondrial MECR that antagonizes influenza virus

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immuno-competitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the pro-viral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the anti-viral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR) that interacts with PB2. cMECR binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and lies solely within cMECR. Thus, alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme.

microbiology↗

bioRxiv2020

Phosphorylation controls RNA binding and transcription by the influenza virus polymerase

The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. We previously showed that phosphorylation regulates genome replication by controlling assembly of the replication machinery (Mondal, et al. 2017). However, it remained unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle.

microbiology↗

bioRxiv2019

EPS8 facilitates uncoating of influenza A virus

All viruses balance interactions between cellular machinery co-opted to support replication and host factors deployed to halt the infection. We used gene correlation analysis to perform an unbiased screen for host factors involved in influenza A virus (FLUAV) infection. Our screen identified the cellular factor epidermal growth factor receptor pathway substrate 8 (EPS8) as the highest confidence pro-viral candidate. Knockout and overexpression of EPS8 confirmed its importance in enhancing FLUAV infection and titers. Loss of EPS8 did not affect virion attachment, uptake, or fusion. Rather, our data show that EPS8 specifically functions during virion uncoating. EPS8 physically associated with incoming virion components, and subsequent nuclear import of released ribonucleoprotein complexes was significantly delayed in the absence of EPS8. Our study identified EPS8 as a host factor important for uncoating, a crucial step of FLUAV infection during which the interface between the virus and host is still being discovered.

microbiology↗

bioRxiv2018

Differential splicing of ANP32A in birds alters its ability to stimulate RNA synthesis by restricted influenza polymerase

Adaptation of viruses to their host can result in specialization and a restricted host range. Species-specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently been identified as a cellular co-factor impacting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation amongst birds differentially impacts viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

microbiology↗

bioRxiv2017

Photocatalytic disinfection of surfaces with copper doped TiO2 nanotube coatings illuminated by ceiling mounted fluorescent lights

High economic burden is associated with foodborne illnesses. Different disinfection methods are therefore employed in food processing industry; such as use of ultraviolet light or usage of surfaces with copper-containing alloys. However, all the disinfection methods currently in use have some shortcomings. Here we show that copper doped TiO2 nanotubes deposited on existing surfaces and illuminated with ceiling mounted fluorescent lights or additional low power light emitting diodes can be employed for an economical and permanent disinfection of surfaces.\n\nWe deposited the nanotubes on various surfaces: polyethylene terephatlate, polystyrene, and aluminum oxide, where they could withstand repeated washings with neutral, alkaline or acidic medium. Here we show that the polymer surfaces coated with the nanotubes and innoculated with 107 bacteria, illuminated with ceiling mounted fluorescent lights retard the growth of Listeria Innocua by up to 99% in seven hours of exposure to the fluorescent lights, compared to a control surface. The disinfection properties of the surfaces depend mainly on the temperature difference of the surface and the dew point, where for maximum effectiveness of the photocatalytic effect the difference should be at least 2.5 degrees celsius.\n\nUsage of one dimensional nanomaterials, such as TiO2 nanotubes, offers a promising low cost alternative to current disinfection methods, since illumination of surfaces with common fluorescent lights is sufficient to photo-excite the nanotubes, which sequentially produce microbicidal hydroxyl radicals. Future use of such surfaces with antibacterial nano-coating and resulting sterilizing effect holds promise for such materials to be used in different environments or in better control of critical control points in food production as well as an improved biosecurity during the food manufacturing process.

bioengineering↗