Viral 2A Research Articles

Fucci2a: A bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice

Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes—there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.

Molecular and structural basis for the roles of hepatitis C virus polymerase NS5B amino acids 15, 223, and 321 in viral replication and drug resistance.

Resistance to the 2'-F-2'-C-methylguanosine monophosphate nucleotide hepatitis C virus (HCV) inhibitors PSI-352938 and PSI-353661 was associated with a combination of amino acid changes (changes of S to G at position 15 [S15G], C223H, and V321I) within the genotype 2a nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase. To understand the role of these residues in viral replication, we examined the effects of single and multiple point mutations on replication fitness and inhibition by a series of nucleotide analog inhibitors. An acidic residue at position 15 reduced replicon fitness, consistent with its proximity to the RNA template. A change of the residue at position 223 to an acidic or large residue reduced replicon fitness, consistent with its proposed proximity to the incoming nucleoside triphosphate (NTP). A change of the residue at position 321 to a charged residue was not tolerated, consistent with its position within a hydrophobic cavity. This triple resistance mutation was specific to both genotype 2a virus and 2'-F-2'-C-methylguanosine inhibitors. A crystal structure of the NS5B S15G/C223H/V321I mutant of the JFH-1 isolate exhibited rearrangement to a conformation potentially consistent with short primer-template RNA binding, which could suggest a mechanism of resistance accomplished through a change in the NS5B conformation, which was better tolerated by genotype 2a virus than by viruses of other genotypes.

Inhibition of enterovirus 71 by adenosine analog NITD008.

Enterovirus 71 (EV71) is a major viral pathogen in China and Southeast Asia. There is no clinically approved vaccine or antiviral therapy for EV71 infection. NITD008, an adenosine analog, is an inhibitor of flavivirus that blocks viral RNA synthesis. Here we report that NITD008 has potent antiviral activity against EV71. In cell culture, the compound inhibits EV71 at a 50% effective concentration of 0.67 μM and a 50% cytotoxic concentration of 119.97 μM. When administered at 5 mg/kg in an EV71 mouse model, the compound reduced viral loads in various organs and completely prevented clinical symptoms and death. To study the antiviral mechanism and drug resistance, we selected escape mutant viruses by culturing EV71 with increasing concentrations of NITD008. Resistance mutations were reproducibly mapped to the viral 3A and 3D polymerase regions. Resistance analysis with recombinant viruses demonstrated that either a 3A or a 3D mutation alone could lead to resistance to NITD008. A combination of both 3A and 3D mutations conferred higher resistance, suggesting a collaborative interplay between the 3A and 3D proteins during viral replication. The resistance results underline the importance of combination therapy required for EV71 treatment. Importance: Human enterovirus 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide, especially in the Asia-Pacific region. Vaccines and antivirals are urgently needed to prevent and treat EV71 infections. In this study, we report the in vitro and in vivo efficacy of NITD008 (an adenosine analog) as an inhibitor of EV71. The efficacy results validated the potential of nucleoside analogs as antiviral drugs for EV71 infections. Mechanistically, we showed that mutations in the viral 3A and 3D polymerases alone or in combination could confer resistance to NITD008. The resistance results suggest an intrinsic interaction between viral proteins 3A and 3D during replication, as well as the importance of combination therapy for the treatment of EV71 infections.

Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

Enterovirus 71 inhibits cellular type I interferon signaling by downregulating JAK1 protein expression.

Enterovirus 71 (EV71) infection can cause severe disease and lead to death in children. Recurring outbreaks of EV71 have been reported in several countries. Interferons (IFNs) have been used for decades to treat several types of viral infection, but have a limited ability to inhibit EV71 replication. Herein, we intend to investigate the mechanisms by which EV71 inhibits the cellular type I IFN response. In this study, MRC-5 (human embryonic lung fibroblast) or RD (human rhabdomyosarcoma) cells were infected with EV71, and then treated with or without IFN-α2b. Cells were harvested and analyzed by flow cytometry to determine the level of IFNAR1. Cell lysis were prepared to detect the levels of STAT1, STAT2, phosphorylated STAT1, phosphorylated STAT2, IFNAR1, JAK1, and TYK2 by Western blotting. The phosphorylation of STAT1 and STAT2 induced by IFN were inhibited without significant downregulation of IFNAR1 in EV71-infected cells. The EV71-induced suppression of STAT1 and STAT2 phosphorylation was not rescued by the protein tyrosine phosphatases inhibitor, and was independent of suppressor of cytokine signaling protein 1/3 levels. The phosphorylation of JAK1 and TYK2 were inhibited accompanied by EV71-induced downregulation of JAK1, which occurred at a post-transcriptional level and was proteasome independent. JAK1 expression did not decrease, and IFN-α-stimulated STAT1 and STAT2 phosphorylation were not blocked in HEK293T cells overexpressing the EV71 viral protein 2A or 3C. This study demonstrates that EV71 inhibits the cellular type I IFN antiviral pathway by downregulating JAK1, while the expression of IFNAR1 does not significantly alter in EV71-infected cells. Additionally, the EV71 viral proteins 2A and 3C do not act as antagonists of cellular type I IFN signaling.

Diverse outcomes of bovine viral diarrhea virus (BVDV) infections in a herd naturally infected during pregnancy

A beef producer purchased pregnant Angus crossbred cows with nursing calves. The purchased cattle, their nursing calves, and subsequent born calves were not initially tested for bovine viral diarrhea virus (BVDV), nor was there a history of vaccination. Bovine viral diarrhea virus subtype 2a was later isolated from an aborted bovine fetus estimated to be 6.5 months gestational age. The fetus had multiple congenital malformations including arthrogryposis, kyphosis, scoliosis, polydactylism, and cardiac overriding aorta. Testing by immunohistochemistry and virus isolation resulted in the detection of potentially persistently infected cattle, including a yearling and a calf born during the same calving season as the aborted fetus. Viruses isolated from the malformed fetus, the yearling, and the calf born during the same calving season were identical. The malformations observed in the aborted fetus were similar to arthrogryposis multiplexa (AM) and contractural arachnodactyly (CA), diseases associated with genetic defects in the Angus breed. The fetus was tested and found negative for the genetic defect linked with AM and CA. This case illustrates that suspect malformations should also be tested for BVDV, and underscores the potential for disease after failed or inadequate biosecurity.

In Silico Identification and Conservation Analysis of B-cell and T-Cell Epitopes of Hepatitis C Virus 3a Genotype Enveloped Glycoprotein 2 From Pakistan: A Step Towards Heterologous Vaccine Design.

Background:Hepatitis C virus (HCV) is known for the eminent global disease burden responsible for encumbering public health. Development of an effective vaccine is the major need of the day; however, several obstacles loom ahead of this objective. One of the major barriers is that as a RNA virus, it mutates rapidly resulting in high sequence divergence and several viral isolates in the world. Theglycoprotein 2 (gpE2) is the primary component of HCV envelope with direct interaction with the host cell surface receptors; it is an indispensable target of neutralizing antibodies and hence, should be a fundamental component of vaccine design.Objectives:This study focused on B-cells and T-cells epitopes prediction in HCV gpE2, particularly in 3a genotype, in Pakistan and identification of the conserved epitopes among various 3a isolates at global level, principally conserved across HCV major genotypes.Materials and Methods:Epitope finding was done by using online available bioinformatics tools including Immune Epitope Database (IEDB), ProPred-I, and ProPred. Conservation of these epitopes was found by aligning selected gpE2 sequences using MultAlin online software and conservancy analysis tool available at IEDB.Results:Many B-cell and T-cell epitopes predicted in gpE2 were found conserved among HCV 3a genotypes whereas few were conserved in other genotypes anticipating these epitopes as potential candidates of producing strong B-cell and T-cell response against HCV 3a and other genotypes.Conclusions:HCV gpE2 is an ideal target for HCV vaccine. Prediction of epitope immunogenicity and characterization on the basis of peptide sequences will be significantly helpful for development of a heterologous vaccine against HCV variants.

Expression of a humanized viral 2A-mediated lux operon efficiently generates autonomous bioluminescence in human cells.

BackgroundExpression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux) cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines.Methodology/Principal FindingsThe lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations.Conclusions/SignificanceOur results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines tested without the induction of cytotoxicity. This autobioluminescent phenotype allowed for repeated interrogation of populations and self-directed control of bioluminescent activation with detection limits and EC50 values similar to traditional reporter systems, making the autobioluminescent cells amenable to automated monitoring and significantly reducing the time and cost required to perform bioluminescent workflows.

Immunogenicity of an inactivated Chinese bovine viral diarrhea virus 1a (BVDV 1a) vaccine cross protects from BVDV 1b infection in young calves

Bovine viral diarrhea virus (BVDV) 1a and 1b strains are the predominant subgenotypes in China. Because of the genetic and antigenic variability among different BVDV strains, a vaccine effective in one region may fail to protect against infections caused by different virus strains in another region. No BVDV vaccine developed with the predominant strains in China are available. In this study, the immunogenicity of an inactivated Chinese BVDV 1a NM01 vaccine strain was evaluated by challenging with a Chinese BVDV 1b JL strain. Ten 2–4-month-old calves were intramuscularly vaccinated with a single dose of the vaccine strain and boosted with same dose three weeks after the first vaccination, with five mock immunized calves serving as a control group. The average titer of neutralization antibody to BVDV 1a and BVDV 1b of immunized calves reached 1:410 and 1:96, respectively, at 21 days post the second vaccination. Twenty-one days post the second vaccination, all calves were challenged with strain JL. The clinical signs, such as the temperature and leukopenia of the immunized calves and viral shedding, were significantly less than the mock immunized calves after challenging with the virulent BVDV 1b strain, indicating that the BVDV 1a vaccine strain elicited efficacious protection against the endemic BVDV 1b strain in China. To the best of our knowledge, this is the first report of an inactivated BVDV vaccine which demonstrated effective cross-protection against BVDV type 1b infection in China.

Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.

Cleavage of DAP5 by coxsackievirus B3 protease 2A causes its nuclear translocation and inhibition of IRES‐containing gene transcription (836.5)

Coxsackievirus B3 (CVB3) is a primary causative agent of viral myocarditis which may progress to dilated cardiomyopathy (DCM), a leading cause of unexpected death in children and young adults. Upon infection, CVB3 hijacks host cellular translation machinery, generating an environment that favors viral replication. Death associated protein 5 (DAP5) is an internal ribosomal entry site (IRES) specific translation initiation factor, utilized by the cell in conditions of stress, differentiation, cell‐cycle and apoptosis. We previously showed that DAP5 is cleaved during CVB3 infection. In this study we aimed to identify the responsible protease and site of cleavage, as well as the functional impact. Our results found that DAP5 was cleaved by viral protease 2A but not by cellular proteases. We further identified the cleavage site at amino acid G434. Upon cleavage, the N‐terminal product could translocate to the nucleus and inhibited transcription of IRES‐containing pro‐survival genes such as Bcl‐2, GRP‐78 and VEGF. This is the first report to reveal the function and mechanism of DAP5 cleavage in regulating viral and host gene expression during CVB3 infection. Moreover, this study may identify key components necessary for facilitating viral pathogenesis and thus provide novel pharmaceutical targets for viral myocarditis.Grant Funding Source: Canadian Institutes of Health Research

A Complex Comprising Phosphatidylinositol 4-Kinase IIIβ, ACBD3, and Aichi Virus Proteins Enhances Phosphatidylinositol 4-Phosphate Synthesis and Is Critical for Formation of the Viral Replication Complex

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornavirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi virus (AiV), a picornavirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754-766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the in vitro kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated. The phosphatidylinositol 4-kinase PI4KB is a host factor required for the replication of certain picornavirus genomes. Aichi virus, a picornavirus belonging to the genus Kobuvirus, forms a complex comprising one of the viral nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB, the Golgi protein ACBD3, and PI4KB to synthesize PI4P at the sites for viral RNA replication. However, the roles of this protein complex in forming the replication complex are unknown. This study showed that virus RNA replication and individual viral proteins enhance the level of cellular PI4P, and suggested that the viral protein/ACBD3/PI4KB complex plays an important role in forming a functional replication complex. Thus, the present study provides a new example of modulation of cellular lipid metabolism by viruses to support the replication of their genomes.

Epstein–Barr virus latent membrane protein-2A alters mitochondrial dynamics promoting cellular migration mediated by Notch signaling pathway

Recently, migration and invasion of breast cancer cells have been linked with dysregulated mitochondrial dynamics. Mitochondria are essential cellular organelles that undergo continuous dynamic cycles of fission and fusion. It has been proposed that a delicate balance between these two processes is important for many pathophysiological outcomes including cancer. Epstein-Barr virus (EBV) is a gamma herpesvirus that is associated with various lymphoid and epithelial malignancies. The viral latent membrane protein 2A (LMP2A) has been shown to increase the invasive ability and induce epithelial-mesenchymal transition in nasopharyngeal carcinoma. Our present study reveals that mitochondrial dynamics also plays a critical role in Epstein-Barr virus-associated epithelial cancers. Our data indicate that viral LMP2A causes an elevated mitochondrial fission in gastric and breast cancer cells, which is manifested by elevated fission protein dynamin-related protein 1 (Drp1). Furthermore, LMP2A-mediated Notch pathway is responsible for this enhanced fission since inhibitors of the pathway decrease the expression of Drp1.

Synthetic Biology Tools for Bioprospecting of Natural Products in Eukaryotes

Filamentous fungi have the capacity to produce a battery of natural products of often unknown function, synthesized by complex metabolic pathways. Unfortunately, most of these pathways appear silent, many in intractable organisms, and their products consequently unidentified. One basic challenge is the difficulty of expressing a biosynthesis pathway for a complex natural product in a heterologous eukaryotic host. Here, we provide a proof-of concept solution to this challenge and describe how the entire penicillin biosynthesis pathway can be expressed in a heterologous host. The method takes advantage of a combination of improved yeast invivo cloning technology, generation of polycistronic mRNA for the gene cluster under study, and an amenable and easily manipulated fungal host, i.e., Aspergillus nidulans. We achieve expression from a single promoter of the pathway genes to yield a large polycistronic mRNA by using viral 2A peptide sequences to direct successful cotranslational cleavage of pathway enzymes.

Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit

Caenorhabditis elegans is a powerful in vivo model in which transgenesis is highly developed. However, while the analysis of biological phenomena often require the expression of more than one protein of interest, no reliable tool exists to ensure efficient concomitant and equivalent expression of more than two polypeptides from a single promoter. We report the use of viral 2A peptides, which trigger a "ribosomal-skip" or "STOP&GO" mechanism during translation, to express multiple proteins from a single vector in C. elegans. Although none of the viruses known to infect C. elegans contain 2A-like sequences, our results show that 2A peptides allow the production of separate functional proteins in all cell types and at all developmental stages tested in the worm. In addition, we constructed a toolkit including a 2A-based polycistronic plasmid and reagents to generate 2A-tagged fosmids. 2A peptides constitute an important tool to ensure the delivery of multiple polypeptides in specific cells, enabling several novel applications such as the reconstitution of multi-subunit complexes.

Evolution of a Cell Culture-Derived Genotype 1a Hepatitis C Virus (H77S.2) during Persistent Infection with Chronic Hepatitis in a Chimpanzee

Persistent infection is a key feature of hepatitis C virus (HCV). However, chimpanzee infections with cell culture-derived viruses (JFH1 or related chimeric viruses that replicate efficiently in cell culture) have been limited to acute-transient infections with no pathogenicity. Here, we report persistent infection with chronic hepatitis in a chimpanzee challenged with cell culture-derived genotype 1a virus (H77S.2) containing 6 cell culture-adaptive mutations. Following acute-transient infection with a chimeric H77/JFH1 virus (HJ3-5), intravenous (i.v.) challenge with 10(6) FFU H77S.2 virus resulted in immediate seroconversion and, following an unusual 4- to 6-week delay, persistent viremia accompanied by alanine aminotransferase (ALT) elevation, intrahepatic innate immune responses, and diffuse hepatopathy. This first persistent infection with cell culture-produced HCV provided a unique opportunity to assess evolution of cell culture-adapted virus in vivo. Synonymous and nonsynonymous nucleotide substitution rates were greatest during the first 8 weeks of infection. Of 6 cell culture-adaptive mutations in H77S.2, Q1067R (NS3) had reverted to Q1067 and S2204I (NS5A) was replaced by T2204 within 8 weeks of infection. By 62 weeks, 4 of 6 mutations had reverted to the wild-type sequence, and all reverted to the wild-type sequence by 194 weeks. The data suggest H77S.2 virus has greater potential for persistence and pathogenicity than JFH1 and demonstrate both the capacity of a nonfit virus to persist for weeks in the liver in the absence of detectable viremia as well as strong selective pressure against cell culture-adaptive mutations in vivo. This study shows that mutations promoting the production of infectious genotype 1a HCV in cell culture have the opposite effect and attenuate replication in the liver of the only fully permissive animal species other than humans. It provides the only example to date of persistent infection in a chimpanzee challenged with cell culture-produced virus and provides novel insight into the forces shaping molecular evolution of that virus during 5 years of persistent infection. It demonstrates that a poorly fit virus can replicate for weeks within the liver in the absence of detectable viremia, an observation that expands current concepts of HCV pathogenesis and that is relevant to relapses observed with direct-acting antiviral therapies.

Inhibition of Hepatitis C Virus Replication by GS-6620, a Potent C -Nucleoside Monophosphate Prodrug

As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 μM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

Enterovirus 2A pro Targets MDA5 and MAVS in Infected Cells

RIG-I-like receptors (RLRs) MDA5 and RIG-I are key players in the innate antiviral response. Upon recognition of viral RNA, they interact with MAVS, eventually inducing type I interferon production. The interferon induction pathway is commonly targeted by viruses. How enteroviruses suppress interferon production is incompletely understood. MDA5 has been suggested to undergo caspase- and proteasome-mediated degradation during poliovirus infection. Additionally, MAVS is reported to be cleaved during infection with coxsackievirus B3 (CVB3) by the CVB3 proteinase 3C(pro), whereas MAVS cleavage by enterovirus 71 has been attributed to 2A(pro). As yet, a detailed examination of the RLR pathway as a whole during any enterovirus infection is lacking. We performed a comprehensive analysis of crucial factors of the RLR pathway, including MDA5, RIG-I, LGP2, MAVS, TBK1, and IRF3, during infection of CVB3, a human enterovirus B (HEV-B) species member. We show that CVB3 inhibits the RLR pathway upstream of TBK1 activation, as demonstrated by limited phosphorylation of TBK1 and a lack of IRF3 phosphorylation. Furthermore, we show that MDA5, MAVS, and RIG-I all undergo proteolytic degradation in CVB3-infected cells through a caspase- and proteasome-independent manner. We convincingly show that MDA5 and MAVS cleavages are both mediated by CVB3 2A(pro), while RIG-I is cleaved by 3C(pro). Moreover, we show that proteinases 2A(pro) and 3C(pro) of poliovirus (HEV-C) and enterovirus 71 (HEV-A) exert the same functions. This study identifies a critical role of 2A(pro) by cleaving MDA5 and MAVS and shows that enteroviruses use a common strategy to counteract the interferon response in infected cells. Human enteroviruses (HEVs) are important pathogens that cause a variety of diseases in humans, including poliomyelitis, hand, foot, and mouth disease, viral meningitis, cardiomyopathy, and more. Like many other viruses, enteroviruses target the host immune pathways to gain replication advantage. The MDA5/MAVS pathway is responsible for recognizing enterovirus infections in the host cell and leads to expression of type I interferons (IFN-I), crucial antiviral signaling molecules. Here we show that three species of HEVs all employ the viral proteinase 2A (2A(pro)) to proteolytically target MDA5 and MAVS, leading to an efficient blockade upstream of IFN-I transcription. These observations suggest that MDA5/MAVS antagonization is an evolutionarily conserved and beneficial mechanism of enteroviruses. Understanding the molecular mechanisms of enterovirus immune evasion strategies will help to develop countermeasures to control infections with these viruses in the future.

Uncleaved Dystrophin Induce Cardiac Myocyte Apoptosis in Coxsackievirus Infected Balb/C Background Mice Heart

It has been previously demonstrated that dystrophin is cleaved in the cardiac myocyte by the viral protease 2A following infection with Coxsackievirus B3 (CVB3). The viral protease 2A mediated cardiomyopathy can be prevented by inhibiting cleavage of dystrophin. However, it is less clear whether uncleaved dysdrophin have other heart protective effect in coxsackievirus infection. To address this, we generated a Balb/C background mouse that had a point mutation in dystrophin that prevents cleavage by protease 2A (KI). We show here that when mice expressing cleavage-resistant dystrophin were infected with CVB3, there was increased cardiac myocyte apoptosis. Bax and Bcl-X L mRNA ratio was significantly increased in KI mice heart compare to wild type mice heart. We found cleavage-resistant dystrophin induced the apoptosis related enzyme capspase-3 and caspase-8 activity. In addition, TUNEL stain was observed many TUNEL positive cardiac myocyte in KI mice heart compare to wild type mice heart (3.7% vs 0.3%). However, zVAD treatment for apoptosis blocking was significantly decreased myocardium damage and fibrosis in KI mice heart. These findings indicated that uncleaved dystrophin may have a critical role in cardiac myocyte viral propagation. Uncleaved dystrophin mutant induced cardiac myocyte apoptosis. It delayed coxsackievirus propagation in cardiac myocyte and could prevent cardiac myocyte death.

The use of an adeno-associated viral vector for efficient bicistronic expression of two genes in the central nervous system.

Recombinant adeno-associated viral (AAV) vectors are one of the most promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). Preclinical testing of novel gene therapies requires the careful design and production of AAV vectors and their successful application in a model of CNS injury. One major limitation of AAV vectors is their limited packaging capacity (<5 kb) making the co-expression of two genes (e.g., from two promoters) difficult. An internal ribosomal entry site has been used to express two genes: However, the second transgene is often expressed at lower levels than the first. In addition to this, achieving high levels of transduction in the CNS can be challenging. In this chapter we describe the cloning of a bicistronic AAV vector that uses the foot-and-mouth disease virus 2A sequence to efficiently express two genes from a single promoter. Bicistronic expression of a therapeutic gene and a reporter gene is desirable so that the axons from transduced neurons can be tracked and, after CNS injury, the amount of axonal sprouting or regeneration quantified. We go on to describe how to perform a pyramidotomy model of CNS injury and the injection of AAV vectors into the sensorimotor cortex to provide efficient transduction and bicistronic gene expression in cortical neurons such that transduced axons are detectable in the dorsal columns of the spinal cord.