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  • Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses

    Zvornicanin, Sarah N; Shaqra, Ala M; Huang, Qiuyu J; Ornelas, Elizabeth; Moghe, Mallika; Knapp, Mark; Moquin, Stephanie; Dovala, Dustin; Schiffer, Celia A; Kurt Yilmaz, Nese (2023-03-18)
    With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 Mpro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 Mpro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.
  • Selection of HIV-1 for resistance to fifth-generation protease inhibitors reveals two independent pathways to high-level resistance

    Spielvogel, Ean; Lee, Sook-Kyung; Zhou, Shuntai; Lockbaum, Gordon J; Henes, Mina; Sondgeroth, Amy; Kosovrasti, Klajdi; Nalivaika, Ellen A; Ali, Akbar; Yilmaz, Nese Kurt; et al. (2023-03-15)
    Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in high drug concentrations that are achieved in vivo. Little is known about the de novo resistance pathway for DRV. We selected for resistance to high drug concentrations against 10 PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small changes in the inhibitor P1'-equivalent position led to preferential use of one pathway over the other. Changes in the inhibitor P2'-equivalent position determined differences in potency that were retained in the resistant viruses and that impacted the selected mutations. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fifth-generation PIs and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.
  • Non-canonical pattern recognition of a pathogen-derived metabolite by a nuclear hormone receptor identifies virulent bacteria in C. elegans

    Peterson, Nicholas D; Tse, Samantha Y; Huang, Qiuyu Judy; Wani, Khursheed A; Schiffer, Celia A; Pukkila-Worley, Read (2023-02-15)
    Distinguishing infectious pathogens from harmless microorganisms is essential for animal health. The mechanisms used to identify infectious microbes are not fully understood, particularly in metazoan hosts that eat bacteria as their food source. Here, we characterized a non-canonical pattern-recognition system in Caenorhabditis elegans (C. elegans) that assesses the relative threat of virulent Pseudomonas aeruginosa (P. Aeruginosa) to activate innate immunity. We discovered that the innate immune response in C. elegans was triggered by phenazine-1-carboxamide (PCN), a toxic metabolite produced by pathogenic strains of P. aeruginosa. We identified the nuclear hormone receptor NHR-86/HNF4 as the PCN sensor in C. elegans and validated that PCN bound to the ligand-binding domain of NHR-86/HNF4. Activation of NHR-86/HNF4 by PCN directly engaged a transcriptional program in intestinal epithelial cells that protected against P. aeruginosa. Thus, a bacterial metabolite is a pattern of pathogenesis surveilled by nematodes to identify a pathogen in its bacterial diet.
  • Structure of the catalytically active APOBEC3G bound to a DNA oligonucleotide inhibitor reveals tetrahedral geometry of the transition state

    Maiti, Atanu; Hedger, Adam K; Myint, Wazo; Balachandran, Vanivilasini; Watts, Jonathan K; Schiffer, Celia A; Matsuo, Hiroshi (2022-11-19)
    APOBEC3 proteins (A3s) are enzymes that catalyze the deamination of cytidine to uridine in single-stranded DNA (ssDNA) substrates, thus playing a key role in innate antiviral immunity. However, the APOBEC3 family has also been linked to many mutational signatures in cancer cells, which has led to an intense interest to develop inhibitors of A3's catalytic activity as therapeutics as well as tools to study A3's biochemistry, structure, and cellular function. Recent studies have shown that ssDNA containing 2'-deoxy-zebularine (dZ-ssDNA) is an inhibitor of A3s such as A3A, A3B, and A3G, although the atomic determinants of this activity have remained unknown. To fill this knowledge gap, we determined a 1.5 Å resolution structure of a dZ-ssDNA inhibitor bound to active A3G. The crystal structure revealed that the activated dZ-H2O mimics the transition state by coordinating the active site Zn2+ and engaging in additional stabilizing interactions, such as the one with the catalytic residue E259. Therefore, this structure allowed us to capture a snapshot of the A3's transition state and suggests that developing transition-state mimicking inhibitors may provide a new opportunity to design more targeted molecules for A3s in the future.
  • Allosteric quinoxaline-based inhibitors of the flavivirus NS2B/NS3 protease

    Zephyr, Jacqueto; Rao, Desaboini Nageswara; Johnson, Colby; Shaqra, Ala M; Nalivaika, Ellen A; Jordan, Aria; Kurt Yilmaz, Nese; Ali, Akbar; Schiffer, Celia A (2022-11-19)
    Viruses from the Flavivirus genus infect millions of people worldwide and cause severe diseases, including recent epidemics of dengue virus (DENV), and Zika virus (ZIKV). There is currently no antiviral treatment against flavivirus infections, despite considerable efforts to develop inhibitors against essential viral enzymes including NS2B/NS3 protease. Targeting the flavivirus NS2B/NS3 protease proved to be challenging because of the conformational dynamics, topology, and electrostatic properties of the active site. Here, we report the identification of quinoxaline-based allosteric inhibitors by fragment-based drug discovery approach as a promising new drug-like scaffold to target the NS2B/NS3 protease. Enzymatic assays and mutational analysis of the allosteric site in ZIKV NS2B/NS3 protease support noncompetitive inhibition mechanism as well as engineered DENV protease construct indicating the compounds likely compete with the NS2B cofactor for binding to the protease domain. Furthermore, antiviral activity confirmed the therapeutic potential of this new inhibitor scaffold.
  • Dual Inhibitors of Main Protease (M) and Cathepsin L as Potent Antivirals against SARS-CoV2

    Mondal, Santanu; Chen, Yongzhi; Lockbaum, Gordon J; Sen, Sudeshna; Chaudhuri, Sauradip; Reyes, Archie C; Lee, Jeong Min; Kaur, Arshia N; Sultana, Nadia; Cameron, Michael D; et al. (2022-11-10)
    Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (MPro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, MPro is a novel therapeutic target. We identified two novel MPro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the MPro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PLPro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC50 values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of MPro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.
  • Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variants

    Li, Qi; Humphries, Fiachra; Girardin, Roxie C; Wallace, Aaron; Ejemel, Monir; Amcheslavsky, Alla; McMahon, Conor T; Schiller, Zachary A; Ma, Zepei; Cruz, John; et al. (2022-09-12)
    Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.
  • Phomoxanthone A Targets ATP Synthase

    Ali, Rameez; Parelkar, Sangram S; Thompson, Paul R; Mitroka-Batsford, Susan; Yerramilli, Siddartha; Scarlata, Suzanne F; Mistretta, Katelyn S; Coburn, Jeannine M; Mattson, Anita E (2022-09-09)
    Phomoxanthone A is a naturally occurring molecule and a powerful anti-cancer agent, although its mechanism of action is unknown. To facilitate the determination of its biological target(s), we used affinity-based labelling using a phomoxanthone A probe. Labelled proteins were pulled down, subjected to chemoproteomics analysis using LC-MS/MS and ATP synthase was identified as a likely target. Mitochondrial ATP synthase was validated in cultured cells lysates and in live intact cells. Our studies show sixty percent inhibition of ATP synthase by 260 μM phomoxanthone A.
  • Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models

    Yusuf, Issa O; Qiao, Tao; Parsi, Sepideh; Tilvawala, Ronak; Thompson, Paul R; Xu, Zuoshang (2022-09-08)
    Increased protein citrullination (PC) and dysregulated protein arginine deiminase (PAD) activity have been observed in several neurodegenerative diseases. PC is a posttranslational modification catalyzed by the PADs. PC converts peptidyl-arginine to peptidyl-citrulline, thereby reducing the positive charges and altering structure and function of proteins. Of the five PADs, PAD2 is the dominant isoform in the central nervous system (CNS). Abnormal PC and PAD dysregulation are associated with numerous pathological conditions, including inflammatory diseases and neurodegeneration. Animal model studies have shown therapeutic efficacy from inhibition of PADs, thus suggesting a role of PC in pathogenesis. To determine whether PC contribute to amyotrophic lateral sclerosis (ALS), a deadly neurodegenerative disease characterized by loss of motor neurons, paralysis, and eventual death, we investigated alterations of PC and PAD2 in two different transgenic mouse models of ALS expressing human mutant SOD1G93A and PFN1C71G, respectively. PC and PAD2 expression are altered dynamically in the spinal cord during disease progression in both models. PC and PAD2 increase progressively in astrocytes with the development of reactive astrogliosis, while decreasing in neurons. Importantly, in the spinal cord white matter, PC accumulates in protein aggregates that contain the myelin proteins PLP and MBP. PC also accumulates progressively in insoluble protein fractions during disease progression. Finally, increased PC and PAD2 expression spatially correlate with areas of the CNS with the most severe motor neuron degeneration. These results suggest that altered PC is an integral part of the neurodegenerative process and potential biomarkers for disease progression in ALS. Moreover, increased PC may contribute to disease-associated processes such as myelin protein aggregation, myelin degeneration, and astrogliosis.
  • Carbamylation of Integrin αIIbβ3: The Mechanistic Link to Platelet Dysfunction in ESKD

    Binder, Veronika; Chruścicka-Smaga, Barbara; Bergum, Brith; Jaisson, Stéphane; Gillery, Philippe; Sivertsen, Joar; Hervig, Tor; Kaminska, Marta; Tilvawala, Ronak; Nemmara, Venkatesh V; et al. (2022-08-29)
    To investigate carbamylation as a potential mechanistic link between uremia and platelet dysfunction in ESKD, we used liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to quantify total homocitrulline, and biotin-conjugated phenylglyoxal labeling and Western blot to detect carbamylated integrin α IIb β 3 (a receptor required for platelet aggregation). Flow cytometry was used to study activation of isolated platelets and platelet-rich plasma. In a transient transfection system, we tested activity and fibrinogen binding of different mutated forms of the receptor. We assessed platelet adhesion and aggregation in microplate assays.
  • The chemical biology of NAD regulation in axon degeneration

    Icso, Janneke; Thompson, Paul R (2022-07-01)
    During axon degeneration, NAD+ levels are largely controlled by two enzymes: nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha and toll interleukin motif containing protein 1 (SARM1). NMNAT2, which catalyzes the formation of NAD+ from NMN and ATP, is actively degraded leading to decreased NAD+ levels. SARM1 activity further decreases the concentration of NAD+ by catalyzing its hydrolysis to form nicotinamide and a mixture of ADPR and cADPR. Notably, SARM1 knockout mice show decreased neurodegeneration in animal models of axon degeneration, highlighting the therapeutic potential of targeting this novel NAD+ hydrolase. This review discusses recent advances in the SARM1 field, including SARM1 structure, regulation, and catalysis as well as the identification of the first SARM1 inhibitors.
  • Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance

    Shaqra, Ala M.; Zvornicanin, Sarah N.; Huang, Qiu Yu J.; Lockbaum, Gordon J.; Knapp, Mark; Tandeske, Laura; Bakan, David T.; Flynn, Julia M.; Bolon, Daniel N.; Moquin, Stephanie; et al. (2022-06-21)
    Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (M(pro)) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 M(pro) bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of M(pro), map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed M(pro) inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.
  • Comprehensive fitness landscape of SARS-CoV-2 M(pro) reveals insights into viral resistance mechanisms

    Flynn, Julia; Samant, Neha S.; Nachum, Gily S.; Bakan, David T.; Yilmaz, Nese Kurt; Schiffer, Celia A.; Moquin, Stephanie A.; Dovala, Dustin; Bolon, Daniel N. (2022-06-20)
    With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs SARS-CoV-2 main protease (M(pro)) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting M(pro) appear promising but will elicit selection pressure for resistance. To understand resistance potential in M(pro), we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high-throughput assays of M(pro) function in yeast, based on either the ability of M(pro) variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to M(pro) function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the M(pro) dimer. The clinical variants of M(pro) were predominantly functional in our screens, indicating that M(pro) is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to M(pro) evolution and that are likely to contribute to drug resistance. This complete mutational guide of M(pro) can be used in the design of inhibitors with reduced potential of evolving viral resistance.
  • HIV-1 VIF and human APOBEC3G interaction directly observed through molecular specific labeling using a new dual promotor vector

    Myint, Wazo; Schiffer, Celia A.; Matsuo, Hiroshi (2022-04-26)
    Over the last few decades, protein NMR isotope labeling methods using E. coli based expression have revolutionized the information accessible from biomolecular NMR experiments. Selective labeling of a protein of interest in a multi-protein complex can significantly reduce the number of cross-peaks and allow for study of large protein complexes. However, limitations still remain since some proteins are not stable independently and cannot be separately labeled in either NMR active isotope enriched or unenriched media and reconstituted into a multimeric complex. To overcome this limitation, the LEGO NMR method was previously developed using protein expression plasmids containing T7 or araBAD promoters to separately express proteins in the same E. coli after changing between labeled and unlabeled media. Building on this, we developed a method to label the Human Immunodeficiency Virus type 1 viral infectivity factor (HIV-1 Vif), a monomerically unstable protein, in complex with CBFbeta, it's host binding partner. We designed a dual promoter plasmid containing both T7 and araBAD promoters to independently control the expression of HIV-1 Vif in NMR active isotope enriched media and CBFbeta in unenriched media. Using this method, we assigned the backbone resonance and directly observed the binding of HIV-1 Vif with APOBEC3G, a host restriction factor to HIV-1.
  • Quantitative structural analysis of influenza virus by cryo-electron tomography and convolutional neural networks

    Huang, Qiu Yu J.; Song, KangKang; Xu, Chen; Bolon, Daniel N.; Wang, Jennifer P.; Finberg, Robert W.; Schiffer, Celia A.; Somasundaran, Mohan (2022-03-14)
    Influenza viruses pose severe public health threats globally. Influenza viruses are extensively pleomorphic, in shape, size, and organization of viral proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships and aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we leveraged convolutional neural networks and cryoET to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza morphology, glycoprotein density, and conducted subtomogram averaging of influenza glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells.
  • Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

    Zephyr, Jacqueto; Desaboini, Nageswara Rao; Vo, Sang V.; Henes, Mina; Kosovrasti, Klajdi; Matthew, Ashley N.; Hedger, Adam; Timm, Jennifer; Chan, Elise T.; Ali, Akbar; et al. (2022-02-17)
    Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.
  • Pathogen infection and cholesterol deficiency activate the C. elegans p38 immune pathway through a TIR-1/SARM1 phase transition

    Peterson, Nicholas D.; Icso, Janneke D.; Salisbury, J. Elizabeth; Thompson, Paul R; Pukkila-Worley, Read (2022-01-31)
    Intracellular signaling regulators can be concentrated into membrane-free, higher ordered protein assemblies to initiate protective responses during stress - a process known as phase transition. Here, we show that a phase transition of the Caenorhabditis elegans Toll/interleukin-1 receptor domain protein (TIR-1), an NAD(+) glycohydrolase homologous to mammalian sterile alpha and TIR motif-containing 1 (SARM1), underlies p38 PMK-1 immune pathway activation in C. elegans intestinal epithelial cells. Through visualization of fluorescently labeled TIR-1/SARM1 protein, we demonstrate that physiologic stresses, both pathogen and non-pathogen, induce multimerization of TIR-1/SARM1 into visible puncta within intestinal epithelial cells. In vitro enzyme kinetic analyses revealed that, like mammalian SARM1, the NAD(+) glycohydrolase activity of C. elegans TIR-1 is dramatically potentiated by protein oligomerization and a phase transition. Accordingly, C. elegans with genetic mutations that specifically block either multimerization or the NAD(+) glycohydrolase activity of TIR-1/SARM1 fail to induce p38 PMK phosphorylation, are unable to increase immune effector expression, and are dramatically susceptible to bacterial infection. Finally, we demonstrate that a loss-of-function mutation in nhr-8, which alters cholesterol metabolism and is used to study conditions of sterol deficiency, causes TIR-1/SARM1 to oligomerize into puncta in intestinal epithelial cells. Cholesterol scarcity increases p38 PMK-1 phosphorylation, primes immune effector induction in a manner that requires TIR-1/SARM1 oligomerization and its intrinsic NAD(+) glycohydrolase activity, and reduces pathogen accumulation in the intestine during a subsequent infection. These data reveal a new adaptive response that allows a metazoan host to anticipate pathogen threats during cholesterol deprivation, a time of relative susceptibility to infection. Thus, a phase transition of TIR-1/SARM1 as a prerequisite for its NAD(+) glycohydrolase activity is strongly conserved across millions of years of evolution and is essential for diverse physiological processes in multiple cell types.
  • Identification of a Permissive Secondary Mutation That Restores the Enzymatic Activity of Oseltamivir Resistance Mutation H275Y

    Jiang, Li; Samant, Neha S.; Liu, Ping; Somasundaran, Mohan; Jensen, Jeffrey D.; Marasco, Wayne A.; Kowalik, Timothy F.; Schiffer, Celia A.; Finberg, Robert W.; Wang, Jennifer P.; et al. (2022-01-19)
    Many oseltamivir resistance mutations exhibit fitness defects in the absence of drug pressure that hinders their propagation in hosts. Secondary permissive mutations can rescue fitness defects and facilitate the segregation of resistance mutations in viral populations. Previous studies have identified a panel of permissive or compensatory mutations in neuraminidase (NA) that restore the growth defect of the predominant oseltamivir resistance mutation (H275Y) in H1N1 influenza A virus. In prior work, we identified a hyperactive mutation (Y276F) that increased NA activity by approximately 70%. While Y276F had not been previously identified as a permissive mutation, we hypothesized that Y276F may counteract the defects caused by H275Y by buffering its reduced NA expression and enzyme activity. In this study, we measured the relative fitness, NA activity, and surface expression, as well as sensitivity to oseltamivir, for several oseltamivir resistance mutations, including H275Y in the wild-type and Y276F genetic background. Our results demonstrate that Y276F selectively rescues the fitness defect of H275Y by restoring its NA surface expression and enzymatic activity, elucidating the local compensatory structural impacts of Y276F on the adjacent H275Y. IMPORTANCE The potential for influenza A virus (IAV) to cause pandemics makes understanding evolutionary mechanisms that impact drug resistance critical for developing surveillance and treatment strategies. Oseltamivir is the most widely used therapeutic strategy to treat IAV infections, but mutations in IAV can lead to drug resistance. The main oseltamivir resistance mutation, H275Y, occurs in the neuraminidase (NA) protein of IAV and reduces drug binding as well as NA function. Here, we identified a new helper mutation, Y276F, that can rescue the functional defects of H275Y and contribute to the evolution of drug resistance in IAV.
  • The role of SERPIN citrullination in thrombosis

    Tilvawala, Ronak; Nemmara, Venkatesh V.; Reyes, Archie C.; Sorvillo, Nicoletta; Salinger, Ari J.; Cherpokova, Deya; Fukui, Saeko; Gutch, Sarah; Wagner, Denisa; Thompson, Paul R (2021-12-16)
    Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.
  • Quantitative Structural Analysis of Influenza Virus by Cryo-electron Tomography and Convolutional Neural Networks [preprint]

    Huang, Qiu Yu; Song, KangKang; Xu, Chen; Bolon, Daniel N.; Wang, Jennifer P.; Finberg, Robert W.; Schiffer, Celia A.; Somasundaran, Mohan (2021-12-09)
    Influenza viruses pose severe public health threats; they cause millions of infections and tens of thousands of deaths annually in the US. Influenza viruses are extensively pleomorphic, in both shape and size as well as organization of viral structural proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships as well as aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and extensive viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we developed a cryoET processing pipeline leveraging convolutional neural networks (CNNs) to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza viral morphology, glycoprotein density, and conduct subtomogram averaging of HA glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells.

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