The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Susan R. Weiss - One of the best experts on this subject based on the ideXlab platform.
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role of the inflammasome related cytokines il 1 and il 18 during infection with Murine Coronavirus
Journal of NeuroVirology, 2017Co-Authors: Zachary Zalinger, Ruth Elliott, Susan R. WeissAbstract:The inflammasome, a cytosolic protein complex that mediates the processing and secretion of pro-inflammatory cytokines, is one of the first responders during viral infection. The cytokines secreted following inflammasome activation, which include IL-1 and IL-18, regulate cells of both the innate and adaptive immune system, guiding the subsequent immune responses. In this study, we used Murine Coronavirus, mouse hepatitis virus (MHV), infection of the central nervous system and liver to assess of the role of the inflammasome and its related cytokines on pathogenesis and host defense during viral infection. Mice lacking all inflammasome signaling due to the absence of caspase-1 and -11 were more vulnerable to infection, with poor survival and elevated viral replication compared to wild-type mice. Mice lacking IL-1 signaling experienced elevated viral replication but similar survival compared to wild-type controls. In the absence of IL-18, mice had elevated viral replication and poor survival, and this protective effect of IL-18 was found to be due to promotion of interferon gamma production in αβ T cells. These data suggest that inflammasome signaling is largely protective during Murine Coronavirus infection, in large part due to the pro-inflammatory effects of IL-18.
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neurovirulent Murine Coronavirus jhm sd uses cellular zinc metalloproteases for virus entry and cell cell fusion
Journal of Virology, 2017Co-Authors: Judith M Phillips, Thomas M. Gallagher, Susan R. WeissAbstract:ABSTRACT The Coronavirus (CoV) S protein requires cleavage by host cell proteases to mediate virus-cell and cell-cell fusion. Many strains of the Murine Coronavirus mouse hepatitis virus (MHV) have distinct, S-dependent organ and tissue tropisms despite using a common receptor, suggesting that they employ different cellular proteases for fusion. In support of this hypothesis, we found that inhibition of endosomal acidification only modestly decreased entry, and overexpression of the cell surface protease TMPRSS2 greatly enhanced entry, of the highly neurovirulent MHV strain JHM.SD relative to their effects on the reference strain, A59. However, TMPRSS2 overexpression decreased MHV structural protein expression, release of infectious particles, and syncytium formation, and endogenous serine protease activity did not contribute greatly to infection. We therefore investigated the importance of other classes of cellular proteases and found that inhibition of matrix metalloproteinase (MMP)- and a disintegrin and metalloprotease (ADAM)-family zinc metalloproteases markedly decreased both entry and cell-cell fusion. Suppression of virus by metalloprotease inhibition varied among tested cell lines and MHV S proteins, suggesting a role for metalloprotease use in strain-dependent tropism. We conclude that zinc metalloproteases must be considered potential contributors to Coronavirus fusion. IMPORTANCE The family Coronaviridae includes viruses that cause two emerging diseases of humans, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), as well as a number of important animal pathogens. Because Coronaviruses depend on host protease-mediated cleavage of their S proteins for entry, a number of protease inhibitors have been proposed as antiviral agents. However, it is unclear which proteases mediate in vivo infection. For example, SARS-CoV infection of cultured cells depends on endosomal acid pH-dependent proteases rather than on the cell surface acid pH-independent serine protease TMPRSS2, but Zhou et al. (Antiviral Res 116:76–84, 2015, doi:10.1016/j.antiviral.2015.01.011) found that a serine protease inhibitor was more protective than a cathepsin inhibitor in SARS-CoV-infected mice. This paper explores the contributions of endosomal acidification and various proteases to Coronavirus infection and identifies an unexpected class of proteases, the matrix metalloproteinase and ADAM families, as potential targets for antiCoronavirus therapy.
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Murine AKAP7 Has a 2=,5=-Phosphodiesterase Domain That Can Complement an Inactive Murine Coronavirus ns2 Gene
2016Co-Authors: Susan R. Weiss, Robert C H. SilvermanaAbstract:ABSTRACT Viral 2=,5=-phosphodiesterases (2=,5=-PDEs) help disparate RNA viruses evade the antiviral activity of interferon (IFN) by degrading 2=,5=-oligoadenylate (2-5A) activators of RNase L. A kinase anchoring proteins (AKAPs) bind the regulatory sub-units of protein kinase A (PKA) to localize and organize cyclic AMP (cAMP) signaling during diverse physiological processes. Amongmore than 43 AKAP isoforms, AKAP7 appears to be unique in its homology to viral 2=,5=-PDEs. Here we show that mouse AKAP7 rapidly degrades 2-5A with kinetics similar to that of Murine Coronavirus (mouse hepatitis virus [MHV]) strain A59 ns2 and human rotavirus strainWAVP3 proteins. To determine whether AKAP7 could substitute for a viral 2=,5=-PDE, we inserted AKAP7 cDNA into anMHV genome with an inactivated ns2 gene. The AKAP7 PDE domain or N-terminally truncated AKAP7 (both lacking a nuclear localizationmotif), but not full-length AKAP7 or a mutant, AKAP7H185R, PDE domain restored the infectivity of ns2 mutant MHV in bone marrowmacrophages and in livers of infected mice. Interestingly, the AKAP7 PDE domain and N-terminally deleted AKAP7 were present in the cytoplasm (the site of MHV replication), whereas full-length AKAP7 was observed only in nuclei. We suggest the possibility that viral acquisition of the host AKAP7 PDE domainmight have occurred during evolution, allowing diverse RNA viruses to antagonize the RNase L pathway. IMPORTANCE Early virus-host interactions determine whether an infection is established, highlighting the need to understand fundamental mechanisms regulating viral pathogenesis. Recently, our laboratories reported a novel mode of regulation of the IFN antiviral response. We showed that the Coronavirus MHV accessory protein ns2 antagonizes the type I IFN response, pro
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Activation of RNase L by Murine Coronavirus in Myeloid Cells Is Dependent on Basal Oas Gene Expression and Independent of Virus-Induced Interferon.
Journal of virology, 2016Co-Authors: L. Dillon Birdwell, Zachary Zalinger, Ruth Elliott, Kristine M Rose, Robert H. Silverman, Patrick W. Wright, Susan R. WeissAbstract:ABSTRACT The oligoadenylate synthetase (OAS)-RNase L pathway is a potent interferon (IFN)-induced antiviral activity. Upon sensing double-stranded RNA, OAS produces 2′,5′-oligoadenylates (2-5A), which activate RNase L. Murine Coronavirus (mouse hepatitis virus [MHV]) nonstructural protein 2 (ns2) is a 2′,5′-phosphodiesterase (PDE) that cleaves 2-5A, thereby antagonizing RNase L activation. PDE activity is required for robust replication in myeloid cells, as a mutant of MHV (ns2 H126R ) encoding an inactive PDE fails to antagonize RNase L activation and replicates poorly in bone marrow-derived macrophages (BMM), while ns2 H126R replicates to high titer in several types of nonmyeloid cells, as well as in IFN receptor-deficient ( Ifnar1 −/− ) BMM. We reported previously that myeloid cells express significantly higher basal levels of OAS transcripts than nonmyeloid cells. Here, we investigated the contributions of Oas gene expression, basal IFN signaling, and virus-induced IFN to RNase L activation. Infection with ns2 H126R activated RNase L in Ifih1 −/− BMM to a similar extent as in wild-type (WT) BMM, despite the lack of IFN induction in the absence of MDA5 expression. However, ns2 H126R failed to induce RNase L activation in BMM treated with IFNAR1-blocking antibody, as well as in Ifnar1 −/− BMM, both expressing low basal levels of Oas genes. Thus, activation of RNase L does not require virus-induced IFN but rather correlates with adequate levels of basal Oas gene expression, maintained by basal IFN signaling. Finally, overexpression of RNase L is not sufficient to compensate for inadequate basal OAS levels. IMPORTANCE The oligoadenylate synthetase (OAS)-RNase L pathway is a potent antiviral activity. Activation of RNase L during Murine Coronavirus (mouse hepatitis virus [MHV]) infection of myeloid cells correlates with high basal Oas gene expression and is independent of virus-induced interferon secretion. Thus, our data suggest that cells with high basal Oas gene expression levels can activate RNase L and thereby inhibit virus replication early in infection upon exposure to viral double-stranded RNA (dsRNA) before the induction of interferon and prior to transcription of interferon-stimulated antiviral genes. These findings challenge the notion that activation of the OAS-RNase L pathway requires virus to induce type I IFN, which in turn upregulates OAS gene expression, as well as to provide dsRNA to activate OAS. Our data further suggest that myeloid cells may serve as sentinels to restrict viral replication, thus protecting other cell types from infection.
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mda5 is critical to host defense during infection with Murine Coronavirus
Journal of Virology, 2015Co-Authors: Zachary Zalinger, Ruth Elliott, Kristine M Rose, Susan R. WeissAbstract:Infection with the Murine Coronavirus mouse hepatitis virus (MHV) activates the pattern recognition receptors melanoma differentiation-associated gene 5 (MDA5) and Toll-like receptor 7 (TLR7) to induce transcription of type I interferon. Type I interferon is crucial for control of viral replication and spread in the natural host, but the specific contributions of MDA5 signaling to this pathway as well as to pathogenesis and subsequent immune responses are largely unknown. In this study, we use MHV infection of the liver as a model to demonstrate that MDA5 signaling is critically important for controlling MHV-induced pathology and regulation of the immune response. Mice deficient in MDA5 expression (MDA5−/− mice) experienced more severe disease following MHV infection, with reduced survival, increased spread of virus to additional sites of infection, and more extensive liver damage than did wild-type mice. Although type I interferon transcription decreased in MDA5−/− mice, the interferon-stimulated gene response remained intact. Cytokine production by innate and adaptive immune cells was largely intact in MDA5−/− mice, but perforin induction by natural killer cells and levels of interferon gamma, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in serum were elevated. These data suggest that MDA5 signaling reduces the severity of MHV-induced disease, at least in part by reducing the intensity of the proinflammatory cytokine response. IMPORTANCE Multicellular organisms employ a wide range of sensors to detect viruses and other pathogens. One such sensor, MDA5, detects viral RNA and triggers induction of type I interferons, chemical messengers that induce inflammation and help regulate the immune responses. In this study, we sought to determine the role of MDA5 during infection with mouse hepatitis virus, a Murine Coronavirus used to model viral hepatitis as well as other human diseases. We found that mice lacking the MDA5 sensor were more susceptible to infection than were mice with MDA5 and experienced decreased survival. Viral replication in the liver was similar in mice with and without MDA5, but liver damage was increased in MDA5−/− mice, suggesting that the immune response is causing the damage. Production of several proinflammatory cytokines was elevated in MDA5−/− mice, suggesting that MDA5 may be responsible for keeping pathological inflammatory responses in check.
Fumihiro Taguchi - One of the best experts on this subject based on the ideXlab platform.
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Receptor-Independent Infection of Murine Coronavirus: Analysis by Spinoculation
Journal of virology, 2006Co-Authors: Rie Watanabe, Shutoku Matsuyama, Fumihiro TaguchiAbstract:A highly neurovirulent Murine Coronavirus JHMV (wild-type [wt] JHMV) is known to spread from cells infected via the Murine Coronavirus mouse hepatitis virus receptor (MHVR) to cells without MHVR (MHVR-independent infection), whereas a mutant virus isolated from wt JHMV, srr7, spread only in an MHVR-dependent fashion. These observations were obtained by the overlay of JHMV-infected cells onto receptor-negative cells that are otherwise resistant to wt JHMV infection. MHVR-independent infection is hypothetically thought to be attributed to a naturally occurring fusion activation of the wt JHMV S protein, which did not occur in the case of srr7. Attachment of S protein on cells without MHVR during the S-protein activation process seems to be a key condition. Thus, in the present study, we tried to see whether wt JHMV virions that are attached on MHVR-negative cells are able to infect those cells. In order to make virions attach to the cell surface without MHVR, we have used spinoculation, namely, the centrifugation of cells together with inoculated virus at 3,000 rpm for 2 h. This procedure forces viruses to attach to the cell surface, as revealed by quantitative estimation of attached virions by real-time PCR and also facilitated wt JHMV infection to MHVR-negative cells, but failed to do so for srr7. Virions of both wt and srr7 attached on MHVR-negative cells by spinoculation were facilitated for infection in the presence of a soluble form of MHVR that induces conformational changes of both wt and srr7. It was further revealed that wt JHMV S1, but not srr7, was released from the cell surface when S protein was expressed on cells. These observations support the hypothesis that attachment of the virion to MHVR-negative cells is a critical step and that a unique feature of wt JHMV S1 to be released from S2 in a naturally occurring event is involved in an MHVR-independent infection.
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Receptor-Induced Conformational Changes of Murine Coronavirus Spike Protein
Journal of virology, 2002Co-Authors: Shutoku Matsuyama, Fumihiro TaguchiAbstract:Although Murine Coronavirus mouse hepatitis virus (MHV) enters cells by virus-cell membrane fusion triggered by its spike (S) protein, it is not well known how the S protein participates in fusion events. We reported that the soluble form of MHV receptor (soMHVR) transformed a nonfusogenic S protein into a fusogenic one (F. Taguchi and S. Matsuyama, J. Virol. 76:950-958, 2002). In the present study, we demonstrate that soMHVR induces the conformational changes of the S protein, as shown by the proteinase digestion test. A cl-2 mutant, srr7, of the MHV JHM virus (JHMV) was digested with proteinase K after treatment with soMHVR, and the resultant S protein was analyzed by Western blotting using monoclonal antibody (MAb) 10G, specific for the membrane-anchored S2 subunit. A 58-kDa fragment, encompassing the two heptad repeats in S2, was detected when srr7 was digested after soMHVR treatment, while no band was seen when the virus was untreated. The appearance of the proteinase-resistant fragment was dependent on the temperature and time of srr7 incubation with soMHVR and also on the concentration of soMHVR. Coimmunoprecipitation indicated that the direct binding of soMHVR to srr7 S protein induced these conformational changes; this was also suggested by the inhibition of the changes following pretreatment of soMHVR with anti-MHVR MAb CC1. soMHVR induced conformational changes of the S proteins of wild-type (wt) JHMV cl-2, as well as revertants from srr7, srr7A and srr7B; however, a major proportion of these S proteins were resistant to proteinase K even without soMHVR treatment. The implications of this proteinase-resistant fraction are discussed. This is the first report on receptor-induced conformational changes of the membrane-anchored fragment of the Coronavirus S protein.
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Unique N-linked glycosylation of Murine Coronavirus MHV-2 membrane protein at the conserved O-linked glycosylation site.
Virus research, 2000Co-Authors: Yasuko K. Yamada, Mikiko Yabe, Takahiro Ohtsuki, Fumihiro TaguchiAbstract:Abstract The membrane (M) proteins of Murine Coronavirus (MHV) strains have been reported to contain only O-linked oligosaccharides. The predicted O-glycosylation site consisting of four amino acid residues of Ser–Ser–Thr–Thr is located immediately adjacent to the initiator Met and is well conserved among MHV strains investigated so far. We analyzed the nucleotide sequence of a highly virulent strain MHV-2 M-coding region and demonstrated that MHV-2 had a unique amino acid, Asn, at position 2 at the conserved O-glycosylation site. We also demonstrated that this substitution added N-linked glycans to MHV-2 M protein resulting in increment of molecular mass of MHV-2 M protein compared with JHM strain having only O-linked glycans.
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Requirement of Proteolytic Cleavage of the Murine Coronavirus MHV-2 Spike Protein for Fusion Activity
Advances in experimental medicine and biology, 1998Co-Authors: Yasuko K. Yamada, Kazuhiro Takimoto, Mikiko Yabe, Fumihiro TaguchiAbstract:The spike (S) protein of a non-fusogenic Murine Coronavirus, MHV-2, was compared to that of a variant, MHV-2f, with fusion activity. Two amino acids differed between the S proteins of these viruses; one was located in the signal sequence (amino acid 12) and the other in the putative cleavage site (amino acid 757). To determine which one of these amino acid changes is important for the alteration of fusogenicity, chimeric S proteins between MHV-2 and -2f were constructed and expressed in DBT cells by a vaccinia virus expression system. The results revealed that one amino acid change (Ser to Arg) at position 757 is responsible for the acquisition of fusogenicity of the MHV-2f S protein. This change also altered the susceptibility to proteolytic cleavage of the MHV-2 S protein which was originally uncleavable. We concluded that the non-fusogenic activity of MHV-2 results from the lack of cleavage of its S protein.
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Isolation and characterization of Murine Coronavirus mutants resistant to neutralization by soluble receptors.
Advances in experimental medicine and biology, 1998Co-Authors: Keiichi Saeki, Nobuhisa Ohtsuka, Fumihiro TaguchiAbstract:Murine Coronavirus mutants resistant to neutralization with soluble receptors were isolated to study the receptor-binding site on the S proteins since such mutants were ex-pected to have mutations in an important site for receptor-binding. We have isolated five soluble receptor-resistant (srr) mutants which had mutations of a single amino acid at 3 different positions in S protein. Srr mutant 11 with an amino acid change at position 65 (Leu to His) in the S1 subunit showed an extremely reduced binding by virus overlay pro-tein blot assay. However srr mutants with a mutation at 1114 (Leu to Phe) (srr mutants 3, 4 and 7) or 1163 (Cys to Phe) (srr mutant 18) in the S2 subunit had receptor-binding activity similar to that of wild type cl-2. These results suggest that an amino acid at position 62 lo-cated in a conserved region among MHV strains is in particular important for receptor binding. We also discuss why srr mutants with a mutation in S2 showed high resistance to neutralization by soluble receptor, irrespective of their binding to MHV receptors.
Kathryn V Holmes - One of the best experts on this subject based on the ideXlab platform.
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Substitutions of conserved amino acids in the receptor-binding domain of the spike glycoprotein affect utilization of Murine CEACAM1a by the Murine Coronavirus MHV-A59.
Virology, 2005Co-Authors: Larissa B. Thackray, Brian C. Turner, Kathryn V HolmesAbstract:The host range of the Murine Coronavirus (MHV) is limited to susceptible mice and Murine cell lines by interactions of the spike glycoprotein (S) with its receptor, mCEACAM1a. We identified five residues in S (S33, L79, T82, Y162 and K183) that are conserved in the receptor-binding domain of MHV strains, but not in related Coronaviruses. We used targeted RNA recombination to generate isogenic viruses that differ from MHV-A59 by amino acid substitutions in S. Viruses with S33R and K183R substitutions had wild type growth, while L79A/T82A viruses formed small plaques. Viruses with S33G, L79M/T82M or K183G substitutions could only be recovered from cells that over-expressed a mutant mCEACAM1a. Viruses with Y162H or Y162Q substitutions were never recovered, while Y162A viruses formed minute plaques. However, viruses with Y162F substitutions had wild type growth, suggesting that Y162 may comprise part of a hydrophobic domain that contacts the MHV-binding site of mCEACAM1a.
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ceacam1a mice are completely resistant to infection by Murine Coronavirus mouse hepatitis virus a59
Journal of Virology, 2004Co-Authors: Erin M. Hemmila, Kathryn V Holmes, Claire Turbide, Melanie Olson, Serge Jothy, Nicole BeaucheminAbstract:CEACAM1a glycoproteins are members of the immunoglobulin (Ig) superfamily and the carcinoembryonic antigen family. Isoforms expressing either two or four alternatively spliced Ig-like domains in mice have been found in a number of epithelial, endothelial, or hematopoietic tissues. CEACAM1a functions as an intercellular adhesion molecule, an angiogenic factor, and a tumor cell growth inhibitor. Moreover, the mouse and human CEACAM1a proteins are targets of viral or bacterial pathogens, respectively, including the Murine Coronavirus mouse hepatitis virus (MHV), Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis, as well as Moraxella catarrhalis in humans. We have shown that targeted disruption of the Ceacam1a (MHVR) gene resulting in a partial ablation of the protein in mice (p/p mice) led to reduced susceptibility to MHV-A59 infection of the modified mice in the BALB/c background. We have now engineered and produced a Ceacam1a−/− mouse that exhibits complete ablation of the CEACAM1a protein in every tissue where it is normally expressed. We report that 3-week-old Ceacam1a−/− mice in the C57BL/6 genetic background are fully resistant to MHV-A59 infection by both intranasal and intracerebral routes. Whereas virus-inoculated wild-type +/+ C57BL/6 mice showed profound liver damage and spinal cord demyelination under these conditions, Ceacam1a−/− mice displayed normal livers and spinal cords. Virus was recovered from liver and spinal cord tissues of +/+ mice but not of −/− mice. These results indicate that CEACAM1a is the sole receptor for MHV-A59 in both liver and brain and that its deletion from the mouse renders the mouse completely resistant to infection by this virus.
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Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the Murine Coronavirus MHV-A59
Virology, 2004Co-Authors: Larissa B. Thackray, Kathryn V HolmesAbstract:The Murine Coronavirus [Murine hepatitis virus (MHV)] is limited to infection of susceptible mice and Murine cell lines by the specificity of the spike glycoprotein (S) for its receptor, Murine carcinoembryonic antigen cell adhesion molecule 1a (mCEACAM1a). We have recently shown that 21 aa substitutions and a 7-aa insert in the N-terminal region of S are associated with the extended host range of a virus variant derived from Murine cells persistently infected with the A59 strain of MHV (MHV-A59). We used targeted RNA recombination (TRR) to generate isogenic viruses that differ from MHV-A59 by the 21 aa substitutions or the 7-aa insert in S. Only viruses with both the 21 aa substitutions and the 7-aa insert in S infected hamster, feline, and monkey cells. These viruses also infected Murine cells in the presence of blocking anti-mCEACAM1a antibodies. Thus, relatively few changes in the N-terminal region of S1 are sufficient to permit MHV-A59 to interact with alternative receptors on Murine and non-Murine cells.
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Receptor-Dependent Coronavirus Infection of Dendritic Cells
Journal of Virology, 2004Co-Authors: Brian C. Turner, Erin M. Hemmila, Nicole Beauchemin, Kathryn V HolmesAbstract:In several mammalian species, including humans, Coronavirus infection can modulate the host immune response. We show a potential role of dendritic cells (DC) in Murine Coronavirus-induced immune modulation and pathogenesis by demonstrating that the JAW SII DC line and primary DC from BALB/c mice and p/p mice with reduced expression of the Murine Coronavirus receptor, Murine CEACAM1a, are susceptible to Murine Coronavirus infection by a receptor-dependent pathway.
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Receptor-Dependent Coronavirus Infection of Dendritic Cells
Journal of Virology, 2004Co-Authors: Brian C. Turner, Erin M. Hemmila, Nicole Beauchemin, Kathryn V HolmesAbstract:In several mammalian species, including humans, Coronavirus infection can modulate the host immune response. We show a potential role of dendritic cells (DC) in Murine Coronavirus-induced immune modulation and pathogenesis by demonstrating that the JAW SII DC line and primary DC from BALB/c mice and p/p mice with reduced expression of the Murine Coronavirus receptor, Murine CEACAM1a, are susceptible to Murine Coronavirus infection by a receptor-dependent pathway.
Susan C. Baker - One of the best experts on this subject based on the ideXlab platform.
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Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus.
Journal of virology, 2020Co-Authors: Xufang Deng, Yafang Chen, Anna M Mielech, Matthew Hackbart, Kristina R Kesely, Robert C Mettelman, Mackenzie E Chapman, Andrew D Mesecar, Amornrat O'brien, Susan C. BakerAbstract:Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a Murine Coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (P < 0.05) in viral titer in liver and spleen at day 5 postinfection (d p.i.), although both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of the PLP2-ubiquitin complex and that PLP2/DUB activity plays a role as an interferon antagonist in Coronavirus pathogenesis.IMPORTANCE Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The Coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant Murine Coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of Coronaviruses.
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structure guided mutagenesis alters deubiquitinating activity 2 and attenuates pathogenesis of a Murine Coronavirus
bioRxiv, 2020Co-Authors: Xufang Deng, Yafang Chen, Anna M Mielech, Matthew Hackbart, Kristina R Kesely, Robert C Mettelman, Amornrat Obrien, Mackenzie E Chapman, Andrew D Mesecar, Susan C. BakerAbstract:Abstract Coronaviruses express a multifunctional papain-like protease, termed PLP2. PLP2 acts as a protease that cleaves the viral replicase polyprotein, and a deubiquitinating (DUB) enzyme which removes ubiquitin moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2 DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a Murine Coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly as the wild-type virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (p Importance Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The Coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (de-conjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin-binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant Murine Coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced pathogenesis in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of Coronaviruses.
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Mutation in Murine Coronavirus replication protein nsp4 alters assembly of double membrane vesicles
Virology, 2008Co-Authors: Mark A. Clementz, Amornrat Kanjanahaluethai, Timothy E. O'brien, Susan C. BakerAbstract:Coronaviruses are positive-strand RNA viruses that replicate in the cytoplasm of infected cells by generating a membrane-associated replicase complex. The replicase complex assembles on double membrane vesicles (DMVs). Here, we studied the role of a putative replicase anchor, nonstructural protein 4 (nsp4), in the assembly of Murine Coronavirus DMVs. We used reverse genetics to generate infectious clone viruses (icv) with an alanine substitution at nsp4 glycosylation site N176 or N237, or an asparagine to threonine substitution (nsp4-N258T), which is proposed to confer a temperature sensitive phenotype. We found that nsp4-N237A is lethal and nsp4-N258T generated a virus (designated Alb ts6 icv) that is temperature sensitive for viral replication. Analysis of Alb ts6 icv-infected cells revealed that there was a dramatic reduction in DMVs and that both nsp4 and nsp3 partially localized to mitochondria when cells were incubated at the non-permissive temperature. These results reveal a critical role of nsp4 in directing Coronavirus DMV assembly.
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Identification of the Murine Coronavirus MP1 cleavage site recognized by papain-like proteinase 2.
Journal of virology, 2003Co-Authors: Amornrat Kanjanahaluethai, Dalia Jukneliene, Susan C. BakerAbstract:The replicase polyprotein of Murine Coronavirus is extensively processed by three proteinases, two papain-like proteinases (PLPs), termed PLP1 and PLP2, and a picornavirus 3C-like proteinase (3CLpro). Previously, we established a trans-cleavage assay and showed that PLP2 cleaves the replicase polyprotein between p210 and membrane protein 1 (MP1) (A. Kanjanahaluethai and S. C. Baker, J. Virol. 74:7911-7921, 2000). Here, we report the results of our studies identifying and characterizing this cleavage site. To determine the approximate position of the cleavage site, we expressed constructs that extended various distances upstream from the previously defined C-terminal end of MP1. We found that the construct extending from the putative PLP2 cleavage site at glycine 2840-alanine 2841 was most similar in size to the processed MP1 replicase product generated in a trans-cleavage assay. To determine which amino acids are critical for PLP2 recognition and processing, we generated 14 constructs with amino acid substitutions upstream and downstream of the putative cleavage site and assessed the effects of the mutations in the PLP2 trans-cleavage assay. We found that substitutions at phenylalanine 2835, glycine 2839, or glycine 2840 resulted in a reduction in cleavage of MP1. Finally, to unequivocally identify this cleavage site, we isolated radiolabeled MP1 protein and determined the position of [35S]methionine residues released by Edman degradation reaction. We found that the amino-terminal residue of MP1 corresponds to alanine 2841. Therefore, Murine Coronavirus PLP2 cleaves the replicase polyprotein between glycine 2840 and alanine 2841, and the critical determinants for PLP2 recognition and processing occupy the P6, P2, and P1 positions of the cleavage site. This study is the first report of the identification and characterization of a cleavage site recognized by Murine Coronavirus PLP2 activity.
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Identification of the polymerase polyprotein products p72 and p65 of the Murine Coronavirus MHV-JHM.
Virus research, 1996Co-Authors: Hong-qiang Gao, Jennifer J. Schiller, Susan C. BakerAbstract:Abstract The RNA polymerase gene of Murine Coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5′-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.
Ralph S. Baric - One of the best experts on this subject based on the ideXlab platform.
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The nsp1, nsp13, and M Proteins Contribute to the Hepatotropism of Murine Coronavirus JHM.WU
Journal of virology, 2015Co-Authors: Rong Zhang, Judith M Phillips, Timothy J Cowley, Ralph S. Baric, Adam D. Steinbrenner, Boyd Yount, Susan R. WeissAbstract:Mouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these full-length cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo. The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-β expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a BetaCoronavirus virulence factor. IMPORTANCE The BetaCoronavirus genus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome Coronavirus (SARS-CoV) and Middle East respiratory syndrome Coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging Coronaviruses, and there are currently no vaccines or effective antivirals for human Coronaviruses. Thus, it is important to understand the virus-host interaction that regulates Coronavirus pathogenesis. Murine Coronavirus infection of mice provides a useful model for the study of Coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in Coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betaCoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies.
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Altered proteolytic processing of the polymerase polyprotein in RNA(-) temperature sensitive mutants of Murine Coronavirus.
Coronaviruses, 1994Co-Authors: Susan C. Baker, Hong-qiang Gao, Ralph S. BaricAbstract:We examined the synthesis and processing of the polymerase polyprotein in RNA(-) temperature sensitive mutant of Murine Coronavirus strain A59. These temperature sensitive mutants of MHV-A59 synthesize viral RNA at the permissive temperature (33.0°C), but are unable to synthesize viral RNA at the nonpermissive temperature (39.5°C). The is mutants have been mapped to five different complementation groups in the polymerase gene. The 5’-most complementation groups, Groups A and B map to a region encoding an autoproteinase responsible for the cleavage of p28, the amino-terminal product of the polymerase polyprotein. We screened six temperature sensitive mutants to determine if there was an alteration in the proteolytic processing of the polymerase polyprotein, particularly in the cleavage of the p28 protein. Two mutants, tsNC9 and tsLA16, had altered proteolytic products at both the permissive and nonpermissive temperatures. One Group B temperature sensitive mutant, designated tsNC11, was defective in the production of p28 protein at the nonpermissive temperature. To further localize the site of the mutation in tsNCl1, RNA representing the 5’-most 5.3 kb region of the polymerase gene was transfected into tsNCllinfected cells and virus production monitored. The transfected RNA was able to complement the defect in tsNC11, resulting in viral RNA synthesis and production of viral particles at the nonpermissive temperature. These results indicate that a gene product from the 5.3 kb region of gene 1 is required for Coronavirus RNA synthesis.
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Altered proteolytic processing of the polymerase polyprotein in RNA(-) temperature sensitive mutants of Murine Coronavirus.
Advances in experimental medicine and biology, 1993Co-Authors: Susan C. Baker, Hong-qiang Gao, Ralph S. BaricAbstract:We examined the synthesis and processing of the polymerase polyprotein in RNA(-) temperature sensitive mutant of Murine Coronavirus strain A59. These temperature sensitive mutants of MHV-A59 synthesize viral RNA at the permissive temperature (33.0 degrees C), but are unable to synthesize viral RNA at the nonpermissive temperature (39.5 degrees C). The ts mutants have been mapped to five different complementation groups in the polymerase gene. The 5'-most complementation groups, Groups A and B, map to a region encoding an autoproteinase responsible for the cleavage of p28, the amino-terminal product of the polymerase polyprotein. We screened six temperature sensitive mutants to determine if there was an alteration in the proteolytic processing of the polymerase polyprotein, particularly in the cleavage of the p28 protein. Two mutants, tsNC9 and tsLA16, had altered proteolytic products at both the permissive and nonpermissive temperatures. One Group B temperature sensitive mutant, designated tsNC11, was defective in the production of p28 protein at the nonpermissive temperature. To further localize the site of the mutation in tsNC11, RNA representing the 5'-most 5.3 kb region of the polymerase gene was transfected into tsNC11-infected cells and virus production monitored. The transfected RNA was able to complement the defect in tsNC11, resulting in viral RNA synthesis and production of viral particles at the nonpermissive temperature. These results indicate that a gene product from the 5.3 kb region of gene 1 is required for Coronavirus RNA synthesis.
