scholarly journals Role of 1’-Ribose Cyano Substitution for Remdesivir to Effectively Inhibit Nucleotide Addition and Proofreading in SARS-CoV-2 Viral RNA Replication

2020 ◽  
Author(s):  
Lu Zhang ◽  
Dong Zhang ◽  
Xiaowei Wang ◽  
Congmin Yuan ◽  
Yongfang Li ◽  
...  
Keyword(s):  
Rational Design ◽  
Molecular Mechanisms ◽  
Cyano Group ◽  
Rna Replication ◽  
Viral Rna ◽  
Upstream Site ◽  
Health Crisis ◽  
Nucleotide Addition

ABSTRACTCOVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising but challenging drug target due to its intrinsic proofreading exoribonuclease (ExoN). Remdesivir targeting SARS-CoV-2 RdRp exerts high drug efficacy in vitro and in vivo. However, its underlying inhibitory mechanisms remain elusive. Here, we performed all-atom molecular dynamics simulations with an accumulated simulation time of 24 microseconds to elucidate the molecular mechanisms underlying the inhibitory effects of Remdesivir. We found that Remdesivir’s 1’-cyano group of possesses the dual role of inhibiting nucleotide addition and proofreading. The presence of its polar 1’-cyano group at an upstream site in RdRp causes instability and hampers RdRp translocation. This leads to a delayed chain termination of RNA extension, which may also subsequently reduce the likelihood for Remdesivir to be cleaved by ExoN acting on the 3’-terminal nucleotide. In addition, our simulations suggest that Remdesivir’s 1’-cyano group can also disrupt the cleavage active site of ExoN via steric interactions, leading to a further reduced cleavage efficiency. Our work provides plausible molecular mechanisms on how Remdesivir inhibits viral RNA replication and may guide rational design for new treatments of COVID-19 targeting viral replication.

scholarly journals Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1α is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication

2007 ◽  
Vol 88 (10) ◽  
pp. 2800-2810 ◽  
Author(s):  
Michela Campagna ◽  
Mauricio Budini ◽  
Francesca Arnoldi ◽  
Ulrich Desselberger ◽  
Jorge E. Allende ◽  
...  
Keyword(s):  
Cytoplasmic Inclusion ◽  
Rna Replication ◽  
Viral Rna ◽  
Structural Protein ◽  
Cytoplasmic Inclusion Bodies ◽  
Replicative Cycle ◽  
In Cells

The rotavirus (RV) non-structural protein 5, NSP5, is encoded by the smallest of the 11 genomic segments and localizes in ‘viroplasms’, cytoplasmic inclusion bodies in which viral RNA replication and packaging take place. NSP5 is essential for the replicative cycle of the virus because, in its absence, viroplasms are not formed and viral RNA replication and transcription do not occur. NSP5 is produced early in infection and undergoes a complex hyperphosphorylation process, leading to the formation of proteins differing in electrophoretic mobility. The role of hyperphosphorylation of NSP5 in the replicative cycle of rotavirus is unknown. Previous in vitro studies have suggested that the cellular kinase CK1α is responsible for the NSP5 hyperphosphorylation process. Here it is shown, by means of specific RNA interference, that in vivo, CK1α is the enzyme that initiates phosphorylation of NSP5. Lack of NSP5 hyperphosphorylation affected neither its interaction with the virus VP1 and NSP2 proteins normally found in viroplasms, nor the production of viral proteins. In contrast, the morphology of viroplasms was altered markedly in cells in which CK1α was depleted and a moderate decrease in the production of double-stranded RNA and infectious virus was observed. These data show that CK1α is the kinase that phosphorylates NSP5 in virus-infected cells and contribute to further understanding of the role of NSP5 in RV infection.

scholarly journals 1’-Ribose Cyano Substitution Allows Remdesivir to Effectively Inhibit Nucleotide Addition and Proofreading during SARS-CoV-2 Viral RNA Replication

2021 ◽  
Author(s):  
Lu Zhang ◽  
Dong Zhang ◽  
Xiaowei Wang ◽  
Congmin Yuan ◽  
Yongfang Li ◽  
...  
Keyword(s):  
Global Health ◽  
Rna Replication ◽  
Interventional Therapy ◽  
Viral Rna ◽  
Effective Inhibitor ◽  
Health Crisis ◽  
Nucleotide Addition

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. Remdesivir is one effective inhibitor for SARS-CoV-2 viral RNA replication. It supersedes other NTP...

scholarly journals Glycine-zipper motifs in hepatitis C virus nonstructural protein 4B are required for the establishment of viral replication organelles

2017 ◽  
pp. JVI.01890-17 ◽  
Author(s):  
David Paul ◽  
Vanesa Madan ◽  
Omar Ramirez ◽  
Maja Bencun ◽  
Ina Karen Stoeck ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Membrane Vesicles ◽  
Rna Replication ◽  
Viral Rna ◽  
Transmembrane Helices ◽  
Double Membrane

Hepatitis C virus (HCV) RNA replication occurs in tight association with remodeled host cell membranes, presenting as cytoplasmic accumulations of single, double and multi membrane vesicles in infected cells. Formation of these so-called replication organelles is mediated by a complex interplay of host cell factors and viral replicase proteins. Of these, nonstructural protein 4B (NS4B), an integral transmembrane protein, appears to play a key role, but little is known about the molecular mechanisms how this protein contributes to organelle biogenesis. Using forward and reverse genetics we identified glycine-zipper motifs within transmembrane helices 2 and 3 of NS4B that are critically involved in viral RNA replication. Foerster resonance energy transfer analysis revealed the importance of the glycine-zippers in NS4B homo and heterotypic self-interactions. Additionally, ultrastructural analysis using electron microscopy unraveled a prominent role of glycine-zipper residues for the subcellular distribution and the morphology of HCV-induced double membrane vesicles. Notably, loss-of-function NS4B glycine-zipper mutants prominently induced single membrane vesicles with secondary invaginations that might represent an arrested intermediate state in double membrane vesicle formation. These findings highlight a so far unknown role of glycine residues within the membrane integral core domain for NS4B self-interaction and functional as well as structural integrity of HCV replication organelles.IMPORTANCERemodeling of the cellular endomembrane system leading to the establishment of replication organelles is a hallmark of positive-strand RNA viruses. In the case of hepatitis C virus (HCV), expression of the nonstructural proteins induces the accumulation of double membrane vesicles that likely arise from a concerted action of viral and co-opted cellular factors. However, the underlying molecular mechanisms are incompletely understood. Here, we identify glycine-zipper motifs within HCV nonstructural protein 4B (NS4B) transmembrane segments 2 and 3 that are crucial for the protein's self-interaction. Moreover, glycine residues within NS4B transmembrane helices critically contribute to the biogenesis of functional replication organelles and thus, efficient viral RNA replication. These results reveal how glycine-zipper motifs in NS4B contribute to structural and functional integrity of the HCV replication organelles and thus, viral RNA replication.

scholarly journals Microfluidic tumor-on-a-chip model to evaluate the role of tumor environmental stress on NK cell exhaustion

2021 ◽  
Vol 7 (8) ◽  
pp. eabc2331 ◽  
Author(s):  
Jose M. Ayuso ◽  
Shujah Rehman ◽  
Maria Virumbrales-Munoz ◽  
Patrick H. McMinn ◽  
Peter Geiger ◽  
...  
Keyword(s):  
Nk Cells ◽  
Immune Cells ◽  
Molecular Mechanisms ◽  
Nk Cell ◽  
Checkpoint Inhibitors ◽  
Waste Product ◽  
Extended Period ◽  
Cell Exhaustion

Solid tumors generate a suppressive environment that imposes an overwhelming burden on the immune system. Nutrient depletion, waste product accumulation, hypoxia, and pH acidification severely compromise the capacity of effector immune cells such as T and natural killer (NK) cells to destroy cancer cells. However, the specific molecular mechanisms driving immune suppression, as well as the capacity of immune cells to adapt to the suppressive environment, are not completely understood. Thus, here, we used an in vitro microfluidic tumor-on-a-chip platform to evaluate how NK cells respond to the tumor-induced suppressive environment. The results demonstrated that the suppressive environment created by the tumor gradually eroded NK cell cytotoxic capacity, leading to compromised NK cell surveillance and tumor tolerance. Further, NK cell exhaustion persisted for an extended period of time after removing NK cells from the microfluidic platform. Last, the addition of checkpoint inhibitors and immunomodulatory agents alleviated NK cell exhaustion.

scholarly journals Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line

2021 ◽  
Vol 22 (3) ◽  
pp. 1163
Author(s):  
Gaia Palmini ◽  
Cecilia Romagnoli ◽  
Simone Donati ◽  
Roberto Zonefrati ◽  
Gianna Galli ◽  
...  
Keyword(s):  
Cell Line ◽  
Molecular Mechanisms ◽  
Osteogenic Sarcoma ◽  
Cancer Cell Line ◽  
Microscopic Features ◽  
In Vitro Establishment ◽  
Profile Characteristic ◽  
First Time

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

scholarly journals Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

2020 ◽  
Vol 10 (1) ◽  
pp. 78
Author(s):  
April Nettesheim ◽  
Myoung Sup Shim ◽  
Angela Dixon ◽  
Urmimala Raychaudhuri ◽  
Haiyan Gong ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Cathepsin B ◽  
Molecular Mechanisms ◽  
Culture Media ◽  
Ecm Remodeling ◽  
Trabecular Meshwork Cells ◽  
Proteolytic Cascade

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

scholarly journals Low Intensity Shockwave Treatment Modulates Macrophage Functions Beneficial to Healing Chronic Wounds

2021 ◽  
Vol 22 (15) ◽  
pp. 7844
Author(s):  
Jason S. Holsapple ◽  
Ben Cooper ◽  
Susan H. Berry ◽  
Aleksandra Staniszewska ◽  
Bruce M. Dickson ◽  
...  
Keyword(s):  
Macrophage Activation ◽  
Molecular Mechanisms ◽  
Chronic Wounds ◽  
Immunohistochemical Analysis ◽  
Therapeutic Effects ◽  
Gene Expressions ◽  
Extracorporeal Shock Wave

Extracorporeal Shock Wave Therapy (ESWT) is used clinically in various disorders including chronic wounds for its pro-angiogenic, proliferative, and anti-inflammatory effects. However, the underlying cellular and molecular mechanisms driving therapeutic effects are not well characterized. Macrophages play a key role in all aspects of healing and their dysfunction results in failure to resolve chronic wounds. We investigated the role of ESWT on macrophage activity in chronic wound punch biopsies from patients with non-healing venous ulcers prior to, and two weeks post-ESWT, and in macrophage cultures treated with clinical shockwave intensities (150–500 impulses, 5 Hz, 0.1 mJ/mm2). Using wound area measurements and histological/immunohistochemical analysis of wound biopsies, we show ESWT enhanced healing of chronic ulcers associated with improved wound angiogenesis (CD31 staining), significantly decreased CD68-positive macrophages per biopsy area and generally increased macrophage activation. Shockwave treatment of macrophages in culture significantly boosted uptake of apoptotic cells, healing-associated cytokine and growth factor gene expressions and modulated macrophage morphology suggestive of macrophage activation, all of which contribute to wound resolution. Macrophage ERK activity was enhanced, suggesting one mechanotransduction pathway driving events. Collectively, these in vitro and in vivo findings reveal shockwaves as important regulators of macrophage functions linked with wound healing. This immunomodulation represents an underappreciated role of clinically applied shockwaves, which could be exploited for other macrophage-mediated disorders.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Perike Srikanth ◽  
Andrielle E Capote ◽  
Alsina Katherina M ◽  
Benjamin Levin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Abstract P458: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atrial Hypocontractility In Atrial Fibrillation

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Srikanth Perike ◽  
Frederick Damen ◽  
Andrielle Capote ◽  
Katherina M Alsina ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Introduction: Atrial fibrillation (AF), is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 2.7 million to 6.1 million and is predictive to increase to 12.1 million in 2030. AF increases the chances of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. Objective: The overexpression of PPP1R12C, causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), decreasing atrial contractility. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluated the role of PP1c-PPP1R12C interaction in MLC2a de-phosphorylation we used Western blots, coimmunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The Overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, that cause a reduction in atrial contractility and increases AF inducibility. All these discoveries advocate that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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