Evasion of Host Innate Immunity by Emerging Viruses: Antagonizing Host RIG-I Pathways

Viruses confront a seemingly dichotomous relationship with their host cells. They must overcome host defenses in order to complete their infectious cycles and generate new viruses yet the host must remain healthy and hospitable for that to take place. Shortly after infection, the RIGI-like receptors (RLRs) within the cytoplasm of the infected cell recognize foreign motifs present in the pathogen. The host responds by activating a signaling pathway that leads to activation of cellular transcription factors, including the NF-κB and interferon regulatory factor 3 (IRF3), that are necessary for induction of the type 1 interferon genes. Many viruses subdue components of the host innate immune system to facilitate viral replication. Viruses with single stranded RNA genomes that possess double stranded replication intermediates, 5’ triphosphates or 5’ diphosphates along with other secondary recognition motifs including length express proteins that either hide their dsRNA from detection by RLRs, interact with RIG-I directly, or interfere with components of the RIG-I pathway with the ultimate goal of evading innate immunity. In every case the end result is that the host antiviral defense system is crippled and viral propagation can proceed. In this review we focus on the eight emerging viruses most likely to cause major epidemics, including Arenaviruses, Bunyaviruses, Coronaviruses, Filoviruses and Paramyxoviruses, as identified by the World Health Organization in 2016. Once fully understood, the mechanisms employed by viruses to evade host cell immunity may serve as effective targets for a variety of antiviral agents

Near-Complete Genome Sequences of Vesicular Stomatitis Virus Indiana Laboratory Strains HR and T1026R1 and Plaque Isolates 22-20 and 22-25

We report four near-complete genome sequences of vesicular stomatitis virus (VSV) Indiana obtained with Sanger and Illumina next-generation sequencing, namely, laboratory strains HR (heat resistant) and T1026R1 and isolates 22-20 and 22-25. Previously, only the M gene of these viruses had been sequenced, and these sequences were not deposited in GenBank

NF-κB Activation Is Delayed in Mouse L929 Cells Infected with Interferon Suppressing, but Not Inducing, Vesicular Stomatitis Virus Strains

AbstractVesicular stomatitis virus (VSV) mutant T1026R1 of the Indiana (IN) serotype is a good inducer of interferon (IFN). This mutant was used to study the activation of NF-κB, a transcription factor necessary for IFN induction, in mouse L929 cells that were stably transfected with a chimeric gene containing the human IFN-β gene promoter attached to the chloramphenicol acetyltransferase (CAT) coding sequence. NF-κB DNA binding activity was detected as early as 30 min after virus adsorption in nuclear extracts, increased up to 4 hr, and then remained constant for at least 6 additional hr. The kinetics of CAT expression correlated with the kinetics of NF-κB nuclear DNA binding activity. Virus entry and delivery of viral components into the cytoplasm were required for NF-κB activation. Exposure of T1026R1 to one hit of UV irradiation nearly completely reduced NF-κB activation. In cells infected with wild-type (wt) VSV (IN), a noninducer of IFN, NF-κB DNA binding activity in the nucleus was delayed for several hours after virus adsorption. Coinfection of wt VSV and T1026R1 resulted in the reduction of T1026R1-promoted NF-κB activation. This inhibitory activity of wt VSV was abolished by one hit of UV irradiation. Under similar conditions expression of the CAT gene was more UV resistant, suggesting that IFN gene expression is regulated at multiple levels

Evolution of drug resistance drives destabilization of flap region dynamics in HIV-1 protease

The HIV-1 protease is one of several common key targets of combination drug therapies for human immunodeficiency virus infection and acquired immunodeficiency syndrome. During the progression of the disease, some individual patients acquire drug resistance due to mutational hotspots on the viral proteins targeted by combination drug therapies. It has recently been discovered that drug-resistant mutations accumulate on the “flap region” of the HIV-1 protease, which is a critical dynamic region involved in nonspecific polypeptide binding during invasion and infection of the host cell. In this study, we utilize machine learning-assisted comparative molecular dynamics, conducted at single amino acid site resolution, to investigate the dynamic changes that occur during functional dimerization and drug binding of wild-type and common drug-resistant versions of the main protease. We also use a multiagent machine learning model to identify conserved dynamics of the HIV-1 main protease that are preserved across simian and feline protease orthologs. We find that a key conserved functional site in the flap region, a solvent-exposed isoleucine (Ile50) that controls flap dynamics is functionally targeted by drug resistance mutations, leading to amplified molecular dynamics affecting the functional ability of the flap region to hold the drugs. We conclude that better long-term patient outcomes may be achieved by designing drugs that target protease regions that are less dependent upon single sites with large functional binding effects

Computational prediction of intracellular targets of wild-type or mutant vesicular stomatitis matrix protein

The matrix (M) protein of vesicular stomatitis virus (VSV) has a complex role in infection and immune evasion, particularly with respect to suppression of Type I interferon (IFN). Viral strains bearing the wild-type (wt) M protein are able to suppress Type I IFN responses. We recently reported that the 22-25 strain of VSV encodes a wt M protein, however its sister plaque isolate, strain 22-20, carries a M[MD52G] mutation that perturbs the ability of the M protein to block NFκB, but not M-mediated inhibition of host transcription. Therefore, although NFκB is activated in 22-20 infected murine L929 cells infected, no IFN mRNA or protein is produced. To investigate the impact of the M[D52G] mutation on immune evasion by VSV, we used transcriptomic data from L929 cells infected with wt, 22-25, or 22-20 to define parameters in a family of executable logical models with the aim of discovering direct targets of viruses encoding a wt or mutant M protein. After several generations of pruning or fixing hypothetical regulatory interactions, we identified specific predicted targets of each strain. We predict that wt and 22-25 VSV both have direct inhibitory actions on key elements of the NFκB signaling pathway, while 22-20 fails to inhibit this pathway

Cardiomyocytes [electronic resource] : Methods and Protocols /

This volume has been assembled for scientists interested in basic and applied biomedical research directed toward understanding the development, genetics, and function of cardiomyocytes.  The methods and protocols contained within it address cell culture techniques, cardiomyocyte differentiation and redifferentiation, experimental induction of cardiomyopathies, introducing genes into cardiomyocytes, genomic approaches to the understanding cardiomyocytes, cryopreservation of neonatal cardiomyocytes, and modeling of cardiomyocyte function. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and current, Cardiomyocytes: Methods and Protocols explores complimentary areas of cardiomyocyte science that, taken together, can inform individuals with a broad range of interests.Generating Primary Cultures of Murine Cardiac Myocytes and Cardiac Fibroblasts to Study Viral Myocarditis -- Enrichment of Cardiomyocytes in Primary Cultures of Murine Neonatal Hearts -- Deep Sequencing of Cardiac MicroRNA-mRNA Interactomes in Clinical and Experimental Cardiomyopathy -- Next Generation Sequencing Technology in the Genetics of Cardiovascular Disease -- Computational Cardiac Electrophysiology: Implementing Mathematical Models of Cardiomyocytes to Simulate Action Potentials of the Heart -- Methods of Myofibrillogenesis Modeling -- Using the Mechanical Bidomain Model to Analyze the Biomechanical Behavior of Cardiomyocytes -- Fabrication of a Myocardial Patch with Cells Differentiated from Human Induced-Pluripotent Stem Cells -- Efficient Differentiation of Cardiomyocytes from Human Pluripotent Stem Cells with Growth Factors -- Isolation, Culturing, and Characterization of Cardiac Muscle Cells from Non-Human Primate Heart Tissue -- Mouse Embryonic Stem Cell-Derived Cardiac Myocytes in a Cell Culture Dish -- Cryopreservation of Neonatal Cardiomyocytes -- Evaluation of Sarcomeric Organization in Human Pluripotent Stem Cell-Derived Cardiomyocytes -- Electrotonic Coupled Metabolic Purification of Chick Cardiomyocytes -- Gene Transfer into Cardiac Myocytes -- Analysis of 4D Myocardial Wall Motion During Early Stages of Chick Heart Development.This volume has been assembled for scientists interested in basic and applied biomedical research directed toward understanding the development, genetics, and function of cardiomyocytes.  The methods and protocols contained within it address cell culture techniques, cardiomyocyte differentiation and redifferentiation, experimental induction of cardiomyopathies, introducing genes into cardiomyocytes, genomic approaches to the understanding cardiomyocytes, cryopreservation of neonatal cardiomyocytes, and modeling of cardiomyocyte function. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and current, Cardiomyocytes: Methods and Protocols explores complimentary areas of cardiomyocyte science that, taken together, can inform individuals with a broad range of interests

The VSV matrix protein inhibits NF-κB and the interferon response independently in mouse L929 cells

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in immune evasion. While VSV has been thought to suppress the interferon (IFN) response primarily by inhibiting host cell transcription and translation, our recent findings indicate that the M protein also targets NF-κB activation. Therefore, the M protein may utilize two distinct mechanisms to limit expression of antiviral genes, inhibiting both host gene expression and NF-κB activation. Here we characterize a recently reported mutation in the M protein [M(D52G)] of VSV isolate 22-20, which suppressed IFN mRNA and protein production despite activating NF-κB. 22-20 inhibited reporter gene expression from multiple promoters, suggesting that 22-20 suppressed the IFN response via M-mediated inhibition of host cell transcription. We propose that suppression of the IFN response and regulation of NF-κB are independent, genetically separable functions of the VSV M protein