Biology subjects

Fehr, A.

Publications and source records attributed to Fehr, A..

10 recordsLinked to original sources

bioRxiv2025

SARS-CoV-2 and MERS-CoV disrupt host protein synthesis via nsp1 with differential effects on the integrated stress response

Coronaviruses pose a serious threat to public health, driving the need for antiviral therapeutics and vaccines. Therefore, it is paramount to understand how this family of viruses evades cellular antiviral responses and establishes productive infection. The conserved coronavirus non-structural protein (nsp)1 has been shown to inhibit host protein synthesis and promote host mRNA degradation while viral mRNAs are protected. We showed previously that SARS-CoV-2 induces activation of host integrated stress response (ISR) kinases PKR and PERK, which promote phosphorylation of eIF2 and consequent inhibition of host protein synthesis. In contrast, eIF2 remains unphosphorylated during MERS-CoV infection. To investigate the interactions of nsp1 and the ISR kinases, we utilized recombinant SARS-CoV-2 and MERS-CoV expressing nsp1 with mutations in each of two conserved domains. Upon infection with SARS-CoV-2 nsp1 mutants, translation was shut down in wildtype (WT) and PKR knockout (KO) cells but rescued in PERK KO cells, likely due to reduced p-eIF2. In contrast, translation was rescued during infection with the analogous MERS-CoV nsp1 mutants even in WT cells. Moreover, SARS-CoV-2 WT suppressed expression of GADD34, a negative regulator of eIF2 phosphorylation, while SARS-CoV-2 nsp1 mutants induced GADD34. In contrast MERS-CoV WT induced GADD34. Utilizing single-molecule fluorescence in situ hybridization, we found that SARS-CoV-2 and MERS-CoV nsp1 promote host mRNA degradation during WT, but not nsp1 mutant, infection. Finally, while SARS-CoV-2 WT suppressed stress granule formation, nsp1 mutants induced stress granules containing host RNA. Thus, SARS-CoV-2 and MERS-CoV differ in interactions with the ISR and nsp1 control of host protein synthesis. SignificanceCoronaviruses cause disease across a wide range of animal species, and the human coronaviruses SARS-CoV-2 and MERS-CoV have caused epidemics of severe respiratory illness. Thus, it is imperative to understand how these viruses antagonize host responses and cause lethal disease. We show here that the betacoronavirus non-structural protein (nsp)1 promotes shutdown of host protein synthesis while preserving viral protein synthesis and, in addition, promotes degradation of host mRNAs. However, SARS-CoV-2 and MERS-CoV differ in their ability to manipulate the host integrated stress response, indicating that it is important to understand detailed coronavirus-host interactions and how they differ even between lethal coronaviruses. Such insights will inform the development of antiviral therapeutics to treat and prevent current and future coronavirus outbreaks.

microbiology↗

bioRxiv2025

An autoimmunity-associated allele of PTPN22 enhances the innate immune response to promote protection against acute coronavirus infection

Allelic variation can impact viral clearance and disease severity. Still, our understanding of the effect of the autoimmunity-associated allelic variant of Ptpn22 (PEP-R619W) on antiviral immunity remains incomplete, as previous reports have only focused on chronic Lymphocytic Choriomeningitis virus (LCMV) infection. This research defines how the loss of Ptpn22 (PEP-null) and PEP-R619W changes antiviral immunity during an acute coronavirus infection. We address the hypothesis that CRISPR/Cas9-generated PEP-null and PEP-R619W mice have enhanced antiviral immunity over PEP-WT mice during coronavirus infection. Following Mouse Hepatitis Virus (MHV) A59 infection, we interrogated pathology, cytokine production, and cellular responses in the blood, spleen, and liver of PEP-WT, PEP-null, and PEP-R619W mice. Key findings show that PEP-R619W mice have reduced viral titer and weight loss, increased survival, and more mature natural killer (NK) cells in the liver and spleen compared to PEP-WT mice. Interestingly, protection against disease in PEP-null mice was inoculation-dose-dependent, whereas PEP-R619W conferred immunity regardless of infection dose. Further, Rag1-/- PEP-R619W mice had increased survival and reduced viral titer over Rag1-/- PEP-WT mice. PEP-R619W mice also had higher concentrations of IFN{gamma} and enhanced IFN{gamma} production by mature NK cells in the liver at 3 days post-infection. Finally, NK cell depletion elevated PEP-R619W viral titer to similar levels as PEP-WT mice. This is one of the first studies investigating the role of Ptpn22 within NK cells and demonstrates that the Ptpn22 allelic variant augments NK cell function and is beneficial during coronavirus infection. Significance StatementApproximately 5-15% of the North American population has the PTPN22 1858C>T allele, which has been linked with numerous autoimmune diseases and is considered the highest non-HLA risk allele for autoimmunity. Due to this link, the PTPN22 1858C>T allele is often considered pathologic or detrimental. However, recent studies have demonstrated the benefit of this allele in protection from chronic virus infection and some cancers. Yet, a significant research gap remains in understanding how the PTPN22 1858C>T allele impacts the immune response during acute, moribund infections. Using mice that are homozygous for the equivalent, autoimmunity-associated allele in Ptpn22, we demonstrate that this allele uniquely augments innate immunity and enhances Natural Killer cell function to protect against coronavirus infection.

immunology↗

bioRxiv2025

Quorum sensing regulation by the nitrogen phosphotransferase system in Pseudomonas aeruginosa

In the opportunistic pathogen Pseudomonas aeruginosa, the nitrogen-related phosphotransferase system (PTSNtr) influences multiple virulence behaviors. The PTSNtr is comprised of three enzymes: first PtsP, then the PtsO phosphocarrier, and the final PtsN phosphoacceptor. We previously showed that ptsP inactivation increases LasI-LasR quorum sensing, a system by which P. aeruginosa regulates genes in response to population density. LasI synthesizes a diffusible autoinducer that binds and activates the LasR receptor, which activates a feedback loop by increasing lasI expression. In this study, we examined the impact of the PTSNtr on quorum sensing. Disruption of ptsP increased the expression of some, but not all, tested quorum-controlled genes, including lasI, phzM (pyocyanin biosynthesis), hcnA (hydrogen cyanide biosynthesis), and, to a lesser extent, rsaL (quorum sensing regulator). Expression of these genes remained dependent on LasR and the autoinducer, whether provided endogenously or exogenously. Increased lasI expression in {Delta}ptsP (or {Delta}ptsO) cells was partly due to the presence of unphosphorylated PtsN, which alone was sufficient to elevate lasI expression. However, we observed residual increases in {Delta}ptsP or {Delta}ptsO cells even in the absence of PtsN, suggesting that PtsP and PtsO can regulate gene expression independent of PtsN. Indeed, genetically disrupting the PtsO phosphorylation site impacted gene expression in the absence of PtsN, and transcriptomic evidence suggested that PtsO and PtsN have distinct regulons. Our results expand our view of how the PTSNtr components function both within and apart from the classic phosphorylation cascade to regulate key virulence behaviors in P. aeruginosa. IMPORTANCEPseudomonas aeruginosa often causes severe and difficult-to-treat infections. P. aeruginosa virulence requires the nitrogen-related phosphotransferase system (PTSNtr), which comprises the phosphocarrier proteins PtsP and PtsO and the final phosphoacceptor, PtsN. The PTSNtr is known to modulate quorum sensing, but little is known about the mechanism of regulation. Here, we examined quorum sensing regulation by the PTSNtr. We showed that the PTSNtr increases quorum sensing-mediated activation of certain genes through the additive effects of both PtsO and PtsN. We also used transcriptomics to determine the regulons of PtsO and PtsN and found that they are largely nonoverlapping. The results position PtsO and PtsN as independent effectors in the Nitro-PTS and shed new light on virulence regulation in this important pathogen.

microbiology↗

bioRxiv2024

PARP14 is pro- and anti-viral host factor that promotes IFN production and affects the replication of multiple viruses

PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-{beta} and IFN-{lambda} responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14s antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1), a DNA virus, vesicular stomatitis virus (VSV), a negative-sense RNA virus, and lymphocytic choriomeningitis virus (LCMV), an ambisense RNA virus, to infect A549 PARP14 knockout (KO) cells. While LCMV infection was unaffected, HSV-1 replication was increased in PARP14 KO cells and VSV replication was decreased. These results indicate that PARP14 restricts HSV-1 replication but enhances the replication of VSV. A PARP14 active site inhibitor had no impact on HSV-1 or VSV replication, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both proviral and antiviral functions targeting multiple viruses. IMPORTANCEThe antiviral response is largely regulated by post-translation modifications (PTM), including ADP-ribosylation. PARP14 is an ADP-ribosyltransferase that is upregulated by interferon and is under positive selection, indicating that it is involved in host-pathogen conflict. However, no anti-viral function has been described for PARP14. Here, we found that PARP14 represses both coronavirus and HSV-1 replication, demonstrating that PARP14 has antiviral functions. Surprisingly, we also found that PARP14 also has pro-viral functions, as it was critical for the efficient replication of VSV. These data indicate that PARP14 has both proviral and antiviral functions. Defining the mechanisms used by PARP14 to both repress and promote virus replication will provide new insights into how PARPs regulate virus infection.

microbiology↗

bioRxiv2023

Discovery of 2-amide-3-methylester thiophenes inhibiting SARS-CoV-2 ADP-ribosyl hydrolysing macrodomain and coronavirus replication

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 {micro}M. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 {micro}M and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of both MHVa prototype CoV, and SARS-CoV-2. Furthermore, sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts.

biochemistry↗

bioRxiv2023

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in mice

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in this set of proteins is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions ({Delta}Mac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV {Delta}Mac1 BACs, SARS-CoV-2 {Delta}Mac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 {Delta}Mac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, {Delta}Mac1 was quickly cleared causing minimal pathology without any morbidity. {Delta}Mac1 SARS-CoV-2 induced increased levels of interferon (IFN) and interferon-stimulated gene (ISG) expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. {Delta}Mac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target. SIGNIFICANCEAll CoVs, including SARS-CoV-2, encode for a conserved macrodomain (Mac1) that counters host ADP-ribosylation. Prior studies with SARS-CoV-1 and MHV found that Mac1 blocks IFN production and promotes CoV pathogenesis, which has prompted the development of SARS-CoV-2 Mac1 inhibitors. However, development of these compounds into antivirals requires that we understand how SARS-CoV-2 lacking Mac1 replicates and causes disease in vitro and in vivo. Here we found that SARS-CoV-2 containing a complete Mac1 deletion replicates normally in cell culture but induces an elevated IFN response, has reduced viral loads in vivo, and does not cause significant disease in mice. These results will provide a roadmap for testing Mac1 inhibitors, help identify Mac1 functions, and open additional avenues for coronavirus therapies.

microbiology↗

bioRxiv2022

Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV-2 nsp3 Macrodomain

A series of amino acid based 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low M Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/482176v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@167aceorg.highwire.dtl.DTLVardef@1d88c47org.highwire.dtl.DTLVardef@1e1b34borg.highwire.dtl.DTLVardef@c2321a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS-CoV is adept at antagonizing early innate immune pathways - interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L) - generated in response to viral double-stranded (ds)RNA generated during genome replication. This is in contrast to SARS-CoV-2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication. ImportanceMiddle East Respiratory Syndrome Coronavirus (MERS-CoV) causes highly lethal respiratory disease. MERS-CoV encodes several innate immune antagonists, accessory proteins NS4a and NS4b unique to the merbeco lineage and the nsp15 protein endoribonuclease (EndoU), conserved among all coronaviruses. While mutation of each antagonist protein alone has little effect on innate immunity, infections with recombinant MERS-CoVs with mutations of EndoU in combination with either NS4a or NS4b, activate innate signaling pathways and are attenuated for replication. Our data indicate that EndoU and accessory proteins NS4a and NS4b together suppress innate immunity during MERS-CoV infection, to optimize viral replication. This is in contrast to SARS-CoV-2 which activates these pathways and consistent with greater mortality observed during MERS-CoV infection compared to SARS-CoV-2.

microbiology↗

bioRxiv2020

β-Coronaviruses use lysosomal organelles for cellular egress.

{beta}-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). While much is known regarding their cellular entry and replication pathways, their mode of egress remains uncertain; however, this is assumed to be via the biosynthetic secretory pathway by analogy to other enveloped viruses. Using imaging methodologies in combination with virus-specific reporters, we demonstrate that {beta}-Coronaviruses utilize lysosomal trafficking for egress from cells. This pathway is regulated by the Arf-like small GTPase Arl8b; thus, virus egress is insensitive to inhibitors of the biosynthetic secretory pathway. Coronavirus infection results in lysosome deacidification, inactivation of lysosomal degradation and disruption of antigen presentation pathways. This coronavirus-induced exploitation of lysosomes provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

microbiology↗

bioRxiv2017

Resolving the Full Spectrum of Human Genome Variation using Linked-Reads

Large-scale population based analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short read whole genome sequencing. However, standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the advantages of short reads. Starting from only [~]1ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as Linked-Reads. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes (Weisenfeld et al. 2017). In this manuscript, we show the advantages of Linked-Reads over standard short read approaches for reference based analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN1 and SMN2. We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

genomics↗