The Experts below are selected from a list of 351 Experts worldwide ranked by ideXlab platform
Cathy L Miller - One of the best experts on this subject based on the ideXlab platform.
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Mammalian Orthoreovirus factories modulate stress granule protein localization by interaction with g3bp1
Journal of Virology, 2017Co-Authors: Promisree Choudhury, Luke D Bussiere, Cathy L MillerAbstract:ABSTRACT Mammalian Orthoreovirus (MRV) infection induces phosphorylation of translation initiation factor eIF2α, which promotes the formation of discrete cytoplasmic inclusions, termed stress granules (SGs). SGs are emerging as a component of the innate immune response to virus infection, and modulation of SG assembly is a common mechanism employed by viruses to counter this antiviral response. We previously showed that MRV infection induces SGs early and then interferes with SG formation as infection proceeds. In this work, we found that SG-associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed virus factories (VFs), where viral transcription, translation, and replication occur. The localization of SG proteins to VFs was dependent on polysome dissociation and occurred via association of the SG effector protein, Ras-GAP SH3-binding protein 1 (G3BP1), with the MRV nonstructural protein σNS, which localizes to VFs via association with VF nucleating protein, μNS. Deletion analysis of the σNS RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed that σNS association and VF localization phenotypes of G3BP1 do not occur solely through RNA or ribosomal binding but require both the RRM and RGG domains of G3BP1 for maximal viral-factory-like structure (VFL) localization and σNS association. Coexpression of σNS and μNS resulted in disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a manner correlating with strain-dependent induction of host translation shutoff. These results suggest that σNS association with G3BP1 and relocalization of G3BP1 to the VF periphery play roles in SG disruption to facilitate MRV replication in the host translational shutoff environment. IMPORTANCE SGs and SG effector proteins have emerged as important, yet poorly understood, players in the host9s innate immune response to virus infection. MRV infection induces SGs early during infection that are dispersed and/or prevented from forming during late stages of infection despite continued activation of the eIF2α signaling pathway. Cellular and viral components involved in disruption of SGs during late stages of MRV infection remain to be elucidated. This work provides evidence that MRV disruption of SGs may be facilitated by association of the MRV nonstructural protein σNS with the major SG effector protein G3BP1 and subsequent localization of G3BP1 and other SG-associated proteins around the peripheries of virus-encoded factories, interrupting the normal formation of SGs. Our findings also reveal the importance of G3BP1 as an inhibitor of MRV replication during infection for the first time.
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Characterization of a replicating Mammalian Orthoreovirus with tetracysteine tagged μNS for live cell visualization of viral factories
Journal of Virology, 2017Co-Authors: Luke D Bussiere, Promisree Choudhury, Bryan H Bellaire, Cathy L MillerAbstract:Within infected host cells, Mammalian Orthoreovirus (MRV) forms viral factories (VFs), which are sites of viral transcription, translation, assembly, and replication. The MRV nonstructural protein μNS comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells, but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6-amino-acid (CCPGCC) tetracysteine (TC) tag and FlAsH-EDT2 reagent. The TC tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live-cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in-depth studies of VF dynamics and host cell interactions.IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, the members of which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection but remain to be fully characterized. To study VFs, researchers have focused on visualizing the nonstructural protein μNS, which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC-labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.
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characterization of a replicating Mammalian Orthoreovirus with tetracysteine tagged μns for live cell visualization of viral factories
bioRxiv, 2017Co-Authors: Luke D Bussiere, Promisree Choudhury, Bryan H Bellaire, Cathy L MillerAbstract:Within infected host cells, Mammalian Orthoreovirus (MRV) forms viral factories (VFs) which are sites of viral transcription, translation, assembly, and replication. MRV non-structural protein, μNS, comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6 amino-acid (CCPGCC) tetracysteine (TC)-tag and FlAsH-EDT2 reagent. The TC-tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC-tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs, and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in depth studies of VF dynamics and host cell interactions.
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Mammalian Orthoreovirus factories modulate stress granule protein localization by interaction with g3bp1
bioRxiv, 2017Co-Authors: Promisree Choudhury, Luke D Bussiere, Cathy L MillerAbstract:Mammalian Orthoreovirus (MRV) infection induces phosphorylation of translation initiation factor eIF2α which promotes formation of discrete cytoplasmic inclusions, termed stress granules (SGs). SGs are emerging as a component of the innate immune response to virus infection, and modulation of SG assembly is a common mechanism employed by viruses to counter this antiviral response. We previously showed that MRV infection induces SGs early, then interferes with SG formation as infection proceeds. In this work, we found that SG associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed virus factories (VFs), where viral transcription, translation, and replication occur. The localization of SG proteins to VFs was dependent on polysome dissociation and occurred via association of SG effector protein, G3BP1, with MRV non-structural protein σNS, which localizes to VFs via association with VF nucleating protein, μNS. Deletion analysis of the σNS RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed that the association and VF localization of G3BP1 is not occurring solely through RNA or ribosomal binding, but requires both RNA and ribosomal binding domains of G3BP1 for maximal VFL localization and σNS association. Co-expression of σNS and μNS resulted in disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a manner correlating with strain-dependent induction of host translation shutoff. These results suggest that σNS association with and relocalization of G3BP1 to the VF periphery plays a role in SG disruption to facilitate MRV replication in the host translational shutoff environment.
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Downregulation of key regulatory proteins in androgen dependent prostate tumor cells by oncolytic reovirus.
Virology, 2015Co-Authors: Pooja Gupta-saraf, Tyler Meseke, Cathy L MillerAbstract:As prostate tumor cell growth depends on hormones, androgen ablation is an effective therapy for prostate cancer (PCa). However, progression of PCa cells to androgen independent growth (castrate resistant prostate cancer, CRPC) results in relapse and mortality. Hypoxia, a microenvironment of low oxygen that modifies the activity of PCa regulatory proteins including the androgen receptor (AR), plays a critical role in progression to CRPC. Therapies targeting hypoxia and the AR may lengthen the time to CRPC progression thereby increasing survival time of PCa patients. Mammalian Orthoreovirus (MRV) has shown promise for the treatment of prostate tumors in vitro and in vivo. In this study, we found that MRV infection induces downregulation of proteins implicated in CRPC progression, interferes with hypoxia-induced AR activity, and induces apoptosis in androgen dependent cells. This suggests MRV possesses traits that could be exploited to create novel therapies for the inhibition of progression to CRPC.
Max L Nibert - One of the best experts on this subject based on the ideXlab platform.
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dissection of Mammalian Orthoreovirus µ2 reveals a self associative domain required for binding to microtubules but not to factory matrix protein µns
PLOS ONE, 2017Co-Authors: Catherine Eichwald, Max L NibertAbstract:Mammalian Orthoreovirus protein μ2 is a component of the viral core particle. Its activities include RNA binding and hydrolysis of the γ-phosphate from NTPs and RNA 5´-termini, suggesting roles as a cofactor for the viral RNA-dependent RNA polymerase, λ3, first enzyme in 5´-capping of viral plus-strand RNAs, and/or prohibitory of RNA-5´-triphosphate-activated antiviral signaling. Within infected cells, μ2 also contributes to viral factories, cytoplasmic structures in which genome replication and particle assembly occur. By associating with both microtubules (MTs) and viral factory matrix protein μNS, μ2 can anchor the factories to MTs, the full effects of which remain unknown. In this study, a protease-hypersensitive region allowed μ2 to be dissected into two large fragments corresponding to residues 1–282 and 283–736. Fusions with enhanced green fluorescent protein revealed that these amino- and carboxyl-terminal regions of μ2 associate in cells with either MTs or μNS, respectively. More exhaustive deletion analysis defined μ2 residues 1–325 as the minimal contiguous region that associates with MTs in the absence of the self-associating tag. A region involved in μ2 self-association was mapped to residues 283–325, and self-association involving this region was essential for MT-association as well. Likewise, we mapped that μNS-binding site in μ2 relates to residues 290–453 which is independent of μ2 self-association. These findings suggest that μ2 monomers or oligomers can bind to MTs and μNS, but that self-association involving μ2 residues 283–325 is specifically relevant for MT-association during viral factories formation.
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localization of Mammalian Orthoreovirus proteins to cytoplasmic factory like structures via nonoverlapping regions of μns
Journal of Virology, 2010Co-Authors: Craig Hastings, Cathy L Miller, Michelle M Arnold, Teresa J Broering, Max L NibertAbstract:Virally induced structures called viral factories form throughout the cytoplasm of cells infected with Mammalian Orthoreoviruses (MRV). When expressed alone in cells, MRV nonstructural protein μNS forms factory-like structures very similar in appearance to viral factories, suggesting that it is involved in forming the structural matrix of these structures. μNS also associates with MRV core particles; the core proteins μ2, λ1, λ2, λ3, and σ2; and the RNA-binding nonstructural protein σNS. These multiple associations result in the recruitment or retention of these viral proteins or particles at factory-like structures. In this study, we identified the regions of μNS necessary and sufficient for these associations and additionally examined the localization of viral RNA synthesis in infected cells. We found that short regions within the amino-terminal 220 residues of μNS are necessary for associations with core particles and necessary and sufficient for associations with the proteins μ2, λ1, λ2, σ2, and σNS. We also found that only the λ3 protein associates with the carboxyl-terminal one-third of μNS and that viral RNA is synthesized within viral factories. These results suggest that μNS may act as a cytoplasmic scaffolding protein involved in localizing and coordinating viral replication or assembly intermediates for the efficient production of progeny core particles during MRV infection.
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Formation of the factory matrix is an important, though not a sufficient function of nonstructural protein μNS during reovirus infection
Virology, 2008Co-Authors: Michelle M Arnold, Kenneth E Murray, Max L NibertAbstract:Abstract Genome replication of Mammalian Orthoreovirus (MRV) occurs in cytoplasmic inclusion bodies called viral factories. Nonstructural protein μNS, encoded by genome segment M3, is a major constituent of these structures. When expressed without other viral proteins, μNS forms cytoplasmic inclusions morphologically similar to factories, suggesting a role for μNS as the factory framework or matrix. In addition, most other MRV proteins, including all five core proteins (λ1, λ2, λ3, μ2, and σ2) and nonstructural protein σNS, can associate with μNS in these structures. In the current study, small interfering RNA targeting M3 was transfected in association with MRV infection and shown to cause a substantial reduction in μNS expression as well as, among other effects, a reduction in infectious yields by as much as 4 log10 values. By also transfecting in vitro-transcribed M3 plus-strand RNA containing silent mutations that render it resistant to the small interfering RNA, we were able to complement μNS expression and to rescue infectious yields by ~ 100-fold. We next used μNS mutants specifically defective at forming factory-matrix structures to show that this function of μNS is important for MRV growth; point mutations in a C-proximal, putative zinc-hook motif as well as small deletions at the extreme C terminus of μNS prevented rescue of viral growth while causing μNS to be diffusely distributed in cells. We furthermore confirmed that an N-terminally truncated form of μNS, designed to represent μNSC and still able to form factory-matrix structures, is unable to rescue MRV growth, localizing one or more other important functions to an N-terminal region of μNS known to be involved in both μ2 and σNS association. Thus, factory-matrix formation is an important, though not a sufficient function of μNS during MRV infection; μNS is multifunctional in the course of viral growth.
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guanidine hydrochloride inhibits Mammalian Orthoreovirus growth by reversibly blocking the synthesis of double stranded rna
Journal of Virology, 2007Co-Authors: Kenneth E Murray, Max L NibertAbstract:Millimolar concentrations of guanidine hydrochloride (GuHCl) are known to inhibit the replication of many plant and animal viruses having positive-sense RNA genomes. For example, GuHCl reversibly interacts with the nucleotide-binding region of poliovirus protein 2CATPase, resulting in a specific inhibition of viral negative-sense RNA synthesis. The use of GuHCl thereby allows for the spatiotemporal separation of poliovirus gene expression and RNA replication and provides a powerful tool to synchronize the initiation of negative-sense RNA synthesis during in vitro replication reactions. In the present study, we examined the effect of GuHCl on Mammalian Orthoreovirus (MRV), a double-stranded RNA (dsRNA) virus from the family Reoviridae. MRV growth in murine L929 cells was reversibly inhibited by 15 mM GuHCl. Furthermore, 15 mM GuHCl provided specific inhibition of viral dsRNA synthesis while sparing both positive-sense RNA synthesis and viral mRNA translation. By using GuHCl to provide temporal separation of MRV gene expression and genome replication, we obtained evidence that MRV primary transcripts support sufficient protein synthesis to assemble morphologically normal viral factories containing functional replicase complexes. In addition, the coordinated use of GuHCl and cycloheximide allowed us to demonstrate that MRV dsRNA synthesis can occur in the absence of ongoing protein synthesis, although to only a limited extent. Future studies utilizing the reversible inhibition of MRV dsRNA synthesis will focus on elucidating the target of GuHCl, as well as the components of the MRV replicase complexes.
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Protective Immunoglobulin A and G Antibodies Bind to Overlapping Intersubunit Epitopes in the Head Domain of Type 1 Reovirus Adhesin σ1
Journal of Virology, 2004Co-Authors: Anna Helander, Cathy L Miller, Kimberly S. Myers, Marian R. Neutra, Max L NibertAbstract:Nonfusogenic Mammalian Orthoreovirus (reovirus) is an enteric pathogen of mice and a useful model for studies of how an enteric virus crosses the mucosal barrier of its host and is subject to control by the mucosal immune system. We recently generated and characterized a new murine immunoglobulin A (IgA)-class monoclonal antibody (MAb), 1E1, that binds to the adhesin fiber, σ1, of reovirus type 1 Lang (T1L) and thereby neutralizes the infectivity of that strain in cell culture. 1E1 is produced in hybridoma cultures as a mixture of monomers, dimers, and higher polymers and is protective against peroral challenges with T1L either when the MAb is passively administered or when it is secreted into the intestines of mice bearing subcutaneous hybridoma tumors. In the present study, selection and analysis of mutants resistant to neutralization by 1E1 identified the region of T1L σ1 to which the MAb binds. The region bound by a previously characterized type 1 σ1-specific neutralizing IgG MAb, 5C6, was identified in the same way. Each of the 15 mutants isolated and analyzed was found to be much less sensitive to neutralization by either 1E1 or 5C6, suggesting the two MAbs bind to largely overlapping regions of σ1. The tested mutants retained the capacity to recognize specific glycoconjugate receptors on rabbit M cells and cultured epithelial cells, even though viral binding to epithelial cells was inhibited by both MAbs. S1 sequence determinations for 12 of the mutants identified σ1 mutations at four positions between residues 415 and 447, which contribute to forming the receptor-binding head domain. When aligned with the σ1 sequence of reovirus type 3 Dearing (T3D) and mapped onto the previously reported crystal structure of the T3D σ1 trimer, the four positions cluster on the side of the σ1 head, across the interface between two subunits. Three such interface-spanning epitopes are thus present per σ1 trimer and require the intact quaternary structure of the head domain for MAb binding. Identification of these intersubunit epitopes on σ1 opens the way for further studies of the mechanisms of antibody-based neutralization and protection with type 1 reoviruses.
Kenneth E Murray - One of the best experts on this subject based on the ideXlab platform.
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the Mammalian Orthoreovirus bicistronic m3 mrna initiates translation using a 5 end dependent scanning mechanism that does not require interaction of 5 3 untranslated regions
Virus Research, 2014Co-Authors: Vidya Sagar, Kenneth E MurrayAbstract:Abstract Mammalian Orthoreovirus mRNAs possess short 5′ UTR, lack 3′ poly(A) tails, and may lack 5′ cap structures at late times post-infection. As such, the mechanisms by which these viral mRNAs recruit ribosomes remain completely unknown. Toward addressing this question, we used bicistronic MRV M3 mRNA to analyze the role of 5′ and 3′ UTRs during MRV protein synthesis. The 5′ UTR was found to be dispensable for translation initiation; however, reducing its length promoted increased downstream initiation. Modifying start site Kozak context altered the ratio of upstream to downstream initiation, whereas mutations in the 3′ UTR did not. Moreover, an M3 mRNA lacking a 3′ UTR was able to rescue MRV infection to WT levels in an siRNA trans-complementation assay. Together, these data allow us to propose a model in which the MRV M3 mRNA initiates translation using a 5′ end-dependent, scanning mechanism that does not require the viral mRNA 3′ UTR or 5′–3′ UTRs interaction.
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The Mammalian Orthoreovirus bicistronic M3 mRNA initiates translation using a 5′ end-dependent, scanning mechanism that does not require interaction of 5′–3′ untranslated regions
Virus Research, 2014Co-Authors: Vidya Sagar, Kenneth E MurrayAbstract:Abstract Mammalian Orthoreovirus mRNAs possess short 5′ UTR, lack 3′ poly(A) tails, and may lack 5′ cap structures at late times post-infection. As such, the mechanisms by which these viral mRNAs recruit ribosomes remain completely unknown. Toward addressing this question, we used bicistronic MRV M3 mRNA to analyze the role of 5′ and 3′ UTRs during MRV protein synthesis. The 5′ UTR was found to be dispensable for translation initiation; however, reducing its length promoted increased downstream initiation. Modifying start site Kozak context altered the ratio of upstream to downstream initiation, whereas mutations in the 3′ UTR did not. Moreover, an M3 mRNA lacking a 3′ UTR was able to rescue MRV infection to WT levels in an siRNA trans-complementation assay. Together, these data allow us to propose a model in which the MRV M3 mRNA initiates translation using a 5′ end-dependent, scanning mechanism that does not require the viral mRNA 3′ UTR or 5′–3′ UTRs interaction.
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Formation of the factory matrix is an important, though not a sufficient function of nonstructural protein μNS during reovirus infection
Virology, 2008Co-Authors: Michelle M Arnold, Kenneth E Murray, Max L NibertAbstract:Abstract Genome replication of Mammalian Orthoreovirus (MRV) occurs in cytoplasmic inclusion bodies called viral factories. Nonstructural protein μNS, encoded by genome segment M3, is a major constituent of these structures. When expressed without other viral proteins, μNS forms cytoplasmic inclusions morphologically similar to factories, suggesting a role for μNS as the factory framework or matrix. In addition, most other MRV proteins, including all five core proteins (λ1, λ2, λ3, μ2, and σ2) and nonstructural protein σNS, can associate with μNS in these structures. In the current study, small interfering RNA targeting M3 was transfected in association with MRV infection and shown to cause a substantial reduction in μNS expression as well as, among other effects, a reduction in infectious yields by as much as 4 log10 values. By also transfecting in vitro-transcribed M3 plus-strand RNA containing silent mutations that render it resistant to the small interfering RNA, we were able to complement μNS expression and to rescue infectious yields by ~ 100-fold. We next used μNS mutants specifically defective at forming factory-matrix structures to show that this function of μNS is important for MRV growth; point mutations in a C-proximal, putative zinc-hook motif as well as small deletions at the extreme C terminus of μNS prevented rescue of viral growth while causing μNS to be diffusely distributed in cells. We furthermore confirmed that an N-terminally truncated form of μNS, designed to represent μNSC and still able to form factory-matrix structures, is unable to rescue MRV growth, localizing one or more other important functions to an N-terminal region of μNS known to be involved in both μ2 and σNS association. Thus, factory-matrix formation is an important, though not a sufficient function of μNS during MRV infection; μNS is multifunctional in the course of viral growth.
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guanidine hydrochloride inhibits Mammalian Orthoreovirus growth by reversibly blocking the synthesis of double stranded rna
Journal of Virology, 2007Co-Authors: Kenneth E Murray, Max L NibertAbstract:Millimolar concentrations of guanidine hydrochloride (GuHCl) are known to inhibit the replication of many plant and animal viruses having positive-sense RNA genomes. For example, GuHCl reversibly interacts with the nucleotide-binding region of poliovirus protein 2CATPase, resulting in a specific inhibition of viral negative-sense RNA synthesis. The use of GuHCl thereby allows for the spatiotemporal separation of poliovirus gene expression and RNA replication and provides a powerful tool to synchronize the initiation of negative-sense RNA synthesis during in vitro replication reactions. In the present study, we examined the effect of GuHCl on Mammalian Orthoreovirus (MRV), a double-stranded RNA (dsRNA) virus from the family Reoviridae. MRV growth in murine L929 cells was reversibly inhibited by 15 mM GuHCl. Furthermore, 15 mM GuHCl provided specific inhibition of viral dsRNA synthesis while sparing both positive-sense RNA synthesis and viral mRNA translation. By using GuHCl to provide temporal separation of MRV gene expression and genome replication, we obtained evidence that MRV primary transcripts support sufficient protein synthesis to assemble morphologically normal viral factories containing functional replicase complexes. In addition, the coordinated use of GuHCl and cycloheximide allowed us to demonstrate that MRV dsRNA synthesis can occur in the absence of ongoing protein synthesis, although to only a limited extent. Future studies utilizing the reversible inhibition of MRV dsRNA synthesis will focus on elucidating the target of GuHCl, as well as the components of the MRV replicase complexes.
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Nucleoside and RNA Triphosphatase Activities of Orthoreovirus Transcriptase Cofactor μ2
Journal of Biological Chemistry, 2003Co-Authors: John S L Parker, Kenneth E Murray, Max L NibertAbstract:Abstract The Mammalian Orthoreovirus (mORV) core particle is an icosahedral multienzyme complex for viral mRNA synthesis and provides a delimited system for mechanistic studies of that process. Previous genetic results have identified the mORV μ 2 protein as a determinant of viral strain differences in the transcriptase and nucleoside triphosphatase activities of cores. New results in this report provided biochemical and genetic evidence that purified μ2 is itself a divalent cation-dependent nucleoside triphosphatase that can remove the 5′ γ-phosphate from RNA as well. Alanine substitutions in a putative nucleotide binding region of μ2 abrogated both functions but did not affect the purification profile of the protein or its known associations with microtubules and mORV μNS protein in vivo. In vitro microtubule binding by purified μ2 was also demonstrated and not affected by the mutations. Purified μ2 was further demonstrated to interact in vitro with the mORV RNA-dependent RNA polymerase, λ3, and the presence of λ3 mildly stimulated the triphosphatase activities of μ2. These findings confirm that μ2 is an enzymatic component of the mORV core and may contribute several possible functions to viral mRNA synthesis.
Bernardo A. Mainou - One of the best experts on this subject based on the ideXlab platform.
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Enhanced Killing of Triple-Negative Breast Cancer Cells by Reassortant Reovirus and Topoisomerase Inhibitors.
Journal of Virology, 2019Co-Authors: Roxana M Rodriguez Stewart, Jameson T.l. Berry, Angela K. Berger, Sung Bo Yoon, Jaime A Guberman, Nirav B. Patel, Gregory K. Tharp, Steven E. Bosinger, Aspen L Hirsch, Bernardo A. MainouAbstract:Breast cancer is the second leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer constitutes a subset of breast cancer that is associated with higher rates of relapse, decreased survival, and limited therapeutic options for patients afflicted with this type of breast cancer. Mammalian Orthoreovirus (reovirus) selectively infects and kills transformed cells, and a serotype 3 reovirus is in clinical trials to assess its efficacy as an oncolytic agent against several cancers. It is unclear if reovirus serotypes differentially infect and kill triple-negative breast cancer cells and if reovirus-induced cytotoxicity of breast cancer cells can be enhanced by modulating the activity of host molecules and pathways. Here, we generated reassortant reoviruses by forward genetics with enhanced infective and cytotoxic properties in triple-negative breast cancer cells. From a high-throughput screen of small-molecule inhibitors, we identified topoisomerase inhibitors as a class of drugs that enhance reovirus infectivity and cytotoxicity of triple-negative breast cancer cells. Treatment of triple-negative breast cancer cells with topoisomerase inhibitors activates DNA damage response pathways, and reovirus infection induces robust production of type III, but not type I, interferon (IFN). Although type I and type III IFNs can activate STAT1 and STAT2, triple-negative breast cancer cellular proliferation is only negatively affected by type I IFN. Together, these data show that reassortant viruses with a novel genetic composition generated by forward genetics in combination with topoisomerase inhibitors more efficiently infect and kill triple-negative breast cancer cells.IMPORTANCE Patients afflicted by triple-negative breast cancer have decreased survival and limited therapeutic options. Reovirus infection results in cell death of a variety of cancers, but it is unknown if different reovirus types lead to triple-negative breast cancer cell death. In this study, we generated two novel reoviruses that more efficiently infect and kill triple-negative breast cancer cells. We show that infection in the presence of DNA-damaging agents enhances infection and triple-negative breast cancer cell killing by reovirus. These data suggest that a combination of a genetically engineered oncolytic reovirus and topoisomerase inhibitors may provide a potent therapeutic option for patients afflicted with triple-negative breast cancer.
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Abstract 2169: Doxorubicin conjugation to reovirus enhances tumor cell-directed oncolysis
Cancer Research, 2019Co-Authors: Jameson T.l. Berry, Bernardo A. MainouAbstract:Breast cancer remains the most diagnosed type of cancer and second leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer (TNBC) constitutes 10-20% of breast cancer cases. TNBCs are characterized by lack of estrogen (ER) and progesterone (PR) receptors and growth factor receptor HER2/Neu on the cell surface. Therapies that target ER, PR, or HER2 are thus ineffective against TNBC. TNBC is primarily treated with cytotoxic chemotherapy or radiation therapy, which can cause significant damage to healthy cells and tissue. Therefore, there is need for therapies that selectively kill TNBC cells with reduced off-tumor effects. The concept of oncolytic virotherapy has existed since the early 1900s. However, oncolytic viruses have limited efficacy as single agents. Mammalian Orthoreovirus (reovirus) is a non-enveloped, segmented double-stranded RNA virus with tropism for transformed cells. Reovirus is in Phase I - III clinical trials to test its oncolytic efficacy against a variety of cancers. Success has been limited and few trials have tested reovirus against breast cancer. In a high-throughput screen of small molecule inhibitors, we identified the topoisomerase II inhibitor doxorubicin hydrochloride (dox) as an enhancer of reovirus infectivity in the TNBC cell line MDA-MB-231. To better control dox delivery and enhance reovirus oncolytic potential, we chemically conjugated dox to reovirus virions (reo-dox). While reo-dox attachment to MDA-MB-231 cells is slightly impaired, viral replication is largely unaffected. Infection of MDA-MB-231 cells with reo-dox resulted in increased cytopathicity and faster induction of cell death than virus alone. MDA-MB-231 cells infected with reo-dox, but not virus alone, induced DNA double-strand breaks and activation of DNA damage response. Together, our findings show that reo-dox exhibits superior toxicity in TNBC cells than virus alone. Future studies will define the mechanism of enhanced cytopathicity of reo-dox and oncolytic efficacy of dox-conjugated reovirus in vivo. Delivery of small molecule inhibitors via conjugation to reovirus particles may provide an effective new method to directly target and kill cancer cells while minimizing toxicity to healthy cells and tissues. Citation Format: Jameson T. Berry, Bernardo A. Mainou. Doxorubicin conjugation to reovirus enhances tumor cell-directed oncolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2169.
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Enhanced Killing of Triple-Negative Breast Cancer Cells by Reassortant Reovirus and Topoisomerase Inhibitors
bioRxiv, 2019Co-Authors: Roxana M Rodriguez Stewart, Jameson T.l. Berry, Angela K. Berger, Sung Bo Yoon, Jaime A Guberman, Nirav B. Patel, Gregory K. Tharp, Steven E. Bosinger, Bernardo A. MainouAbstract:Breast cancer is the second-leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer constitutes a subset of breast cancer that is associated with higher rates of relapse, decreased survival, and limited therapeutic options for patients afflicted with this type of breast cancer. Mammalian Orthoreovirus (reovirus) selectively infects and kills transformed cells and a serotype 3 reovirus is in clinical trials to assess its efficacy as an oncolytic agent. It is unclear if reovirus serotypes differentially infect and kill triple-negative breast cancer cells and whether addition of small molecule inhibitors enhances reovirus-induced cytotoxicity of breast cancer cells. Here, we generate reassortant reoviruses by forward genetics that infect and kill triple-negative breast cancer cells more efficiently than parental viruses. From a high-throughput screen of small molecule inhibitors, we identified topoisomerase inhibitors as drugs that enhance reovirus infectivity and cytotoxicity of triple-negative breast cancer cells. Treatment of cells with topoisomerase inhibitors activates the DNA damage response and infection with reovirus induces robust production of Type III, but not Type I, interferon. Together, data presented show that reassortant viruses generated by forward genetics in combination with topoisomerase inhibitors more efficiently infect and kill triple-negative breast cancer cells.
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Current understanding of reovirus oncolysis mechanisms
Oncolytic virotherapy, 2018Co-Authors: Matthew B. Phillips, Johnasha D. Stuart, Bernardo A. Mainou, Roxana M Rodriguez Stewart, Jameson T.l. Berry, Karl W. BoehmeAbstract:Mammalian Orthoreovirus (reovirus) is under development as a cancer virotherapy. Clinical trials demonstrate that reovirus-based therapies are safe and tolerated in patients with a wide variety of cancers. Although reovirus monotherapy has proven largely ineffective, reovirus sensitizes cancer cells to existing chemotherapeutic agents and radiation. Clinical trials are underway to test the efficacy of reovirus in combination with chemotherapeutic and radiation regimens and to evaluate the effectiveness of reovirus in conjunction with immunotherapies. Central to the use of reovirus to treat cancer is its capacity to directly kill cancer cells and alter the cellular environment to augment other therapies. Apoptotic cell death is a prominent mechanism of reovirus cancer cell killing. However, reoviruses can also kill cancer cells through nonapoptotic mechanisms. Here, we describe mechanisms of reovirus cancer cell killing, highlight how reovirus is used in combination with existing cancer treatments, and discuss what is known as to how reovirus modulates cancer immunotherapy.
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Bacteria and bacterial envelope components enhance Mammalian reovirus thermostability
PLOS Pathogens, 2017Co-Authors: Angela K. Berger, Hong Yi, Daniel B. Kearns, Bernardo A. MainouAbstract:Enteric viruses encounter diverse environments as they migrate through the gastrointestinal tract to infect their hosts. The interaction of eukaryotic viruses with members of the host microbiota can greatly impact various aspects of virus biology, including the efficiency with which viruses can infect their hosts. Mammalian Orthoreovirus, a human enteric virus that infects most humans during childhood, is negatively affected by antibiotic treatment prior to infection. However, it is not known how components of the host microbiota affect reovirus infectivity. In this study, we show that reovirus virions directly interact with Gram positive and Gram negative bacteria. Reovirus interaction with bacterial cells conveys enhanced virion thermostability that translates into enhanced attachment and infection of cells following an environmental insult. Enhanced virion thermostability was also conveyed by bacterial envelope components lipopolysaccharide (LPS) and peptidoglycan (PG). Lipoteichoic acid and N-acetylglucosamine-containing polysaccharides enhanced virion stability in a serotype-dependent manner. LPS and PG also enhanced the thermostability of an intermediate reovirus particle (ISVP) that is associated with primary infection in the gut. Although LPS and PG alter reovirus thermostability, these bacterial envelope components did not affect reovirus utilization of its proteinaceous cellular receptor junctional adhesion molecule-A or cell entry kinetics. LPS and PG also did not affect the overall number of reovirus capsid proteins σ1 and σ3, suggesting their effect on virion thermostability is not mediated through altering the overall number of major capsid proteins on the virus. Incubation of reovirus with LPS and PG did not significantly affect the neutralizing efficiency of reovirus-specific antibodies. These data suggest that bacteria enhance reovirus infection of the intestinal tract by enhancing the thermal stability of the reovirus particle at a variety of temperatures through interactions between the viral particle and bacterial envelope components.
Liandong Qu - One of the best experts on this subject based on the ideXlab platform.
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characterization and pathogenicity of a novel Mammalian Orthoreovirus from wild short nosed fruit bats
Infection Genetics and Evolution, 2016Co-Authors: Zhijie Li, Yuhao Shao, Jin Tian, Xiaozhan Zhang, Xiaoliang Hu, Liandong QuAbstract:Abstract Mammalian Orthoreoviruses (MRVs) have a wide range of geographic distribution and have been isolated from humans and various animals. This study describes the isolation, molecular characterization and analysis of pathogenicity of MRV variant B/03 from wild short-nosed fruit bats. Negative stain electron microscopy illustrated that the B/03 strain is a non-enveloped icosahedral virus with a diameter of 70 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) migration patterns showed that the B/03 viral genome contains 10 segments in a 3:3:4 arrangement. The isolate belongs to MRV serotype 1 based on S1 gene nucleotide sequence data. BALB/c mice experimentally infected with B/03 virus by intranasal inoculation developed severe respiratory distress with tissue damage and inflammation. Lastly, B/03 virus has an increased transmission risk between bats and humans or animals.
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isolation and pathogenicity of the Mammalian Orthoreovirus mpc 04 from masked civet cats
Infection Genetics and Evolution, 2015Co-Authors: Zhijie Li, Yuhao Shao, Jin Tian, Xiaozhan Zhang, Liandong QuAbstract:Mammalian reoviruses (MRVs) are associated with pulmonary infections and have been isolated from humans and various animals experiencing respiratory illness. We report here the first case of an MRV detected in the masked palm civet, which showed the highest similarity to the serotype 3 MRV. Reovirus particles were identified by electron microscopic examination of both negative-stain and thin-section. Genomic pattern analysis on SDS-PAGE showed that MPC/04 had 10-segmented double-strand RNA genome. Intranasal infection of four-week-old female BALB/c mice resulted in fatal respiratory distress but not other routes. Infections caused tissue damage and inflammation. MPC/04 grew to higher titers in the lungs than in other tissues. This research strongly suggests a need for additional experimentation to understand the pathogenic mechanisms of Mammalian Orthoreoviruses in infected animals and humans.
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Isolation and pathogenicity of the Mammalian Orthoreovirus MPC/04 from masked civet cats.
Infection Genetics and Evolution, 2015Co-Authors: Zhijie Li, Yuhao Shao, Jin Tian, Xiaozhan Zhang, Liandong QuAbstract:Mammalian reoviruses (MRVs) are associated with pulmonary infections and have been isolated from humans and various animals experiencing respiratory illness. We report here the first case of an MRV detected in the masked palm civet, which showed the highest similarity to the serotype 3 MRV. Reovirus particles were identified by electron microscopic examination of both negative-stain and thin-section. Genomic pattern analysis on SDS-PAGE showed that MPC/04 had 10-segmented double-strand RNA genome. Intranasal infection of four-week-old female BALB/c mice resulted in fatal respiratory distress but not other routes. Infections caused tissue damage and inflammation. MPC/04 grew to higher titers in the lungs than in other tissues. This research strongly suggests a need for additional experimentation to understand the pathogenic mechanisms of Mammalian Orthoreoviruses in infected animals and humans.
