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Biomedical subjects

Michael Gale

Publications and source records attributed to Michael Gale.

36 records · Page 2Linked to original sources
PubMed2005

Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF.

Toll-like receptors (TLRs) bind pathogen-specific ligands early in infection, initiating signaling pathways that lead to expression of multiple protective cellular genes. Many viruses have evolved strategies that block the effector mechanisms induced through these signaling pathways, but viral interference with critical proximal receptor interactions has not been described. We show here that the NS3/4A serine protease of hepatitis C virus (HCV), a virus notorious for its ability to establish persistent intrahepatic infection, causes specific proteolysis of Toll-IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF or TICAM-1), an adaptor protein linking TLR3 to kinases responsible for activating IFN regulatory factor 3 (IRF-3) and NF-kappaB, transcription factors controlling a multiplicity of antiviral defenses. NS3/4A-mediated cleavage of TRIF reduces its abundance and inhibits polyI:C-activated signaling through the TLR3 pathway before its bifurcation to IRF-3 and NF-kappaB. This uniquely broad mechanism of immune evasion potentially limits expression of multiple host defense genes, thereby promoting persistent infections with this medically important virus.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

The oncogenic potential of hepatitis C virus NS5A sequence variants is associated with PKR regulation.

The NS5A protein of hepatitis C virus (HCV) confers cell growth regulation and has been implicated in viral oncogenesis. Here, we investigated whether highly divergent NS5A proteins obtained from HCV-infected patients presented an oncogenic potential when expressed in mammalian cells. In general, NS5A expression was associated with increased rates of cell growth and culture proliferation. Immortalized primary hepatocyte and immortalized fibroblast cell lines expressing a subset of these sequences exhibited a significant increase in protein synthetic rate, culture saturation density, and a transformed cellular phenotype, as shown by anchorage-independent cell growth and colony formation in soft agar assays. Oncogenic transformation correlated with inhibition of protein kinase R (PKR) activity and concomitant reduction of eukaryotic initiation factor 2alpha (elF2alpha) phosphorylation levels that caused stimulation of mRNA translation. The extent of sequence variation throughout NS5A or within the previously characterized PKR-binding domain was not a predictive indicator of this cellular phenotype, suggesting that sequences outside this region contribute to PKR regulation. Our data indicate that NS5A oncogenic potential is conditional through viral sequence variation. These results provide further evidence to define the PKR pathway as a mediator of cell growth control and suggest that viral regulation of PKR may contribute to hepatocyte growth deregulation during chronic HCV infection.

Amino Acid Sequence↗
PubMed2005

GB virus type C NS5A sequence polymorphisms: association with interferon susceptibility and inhibition of PKR-mediated eIF2alpha phosphorylation.

GB virus type C (GBV-C) causes persistent infection in humans, although the mechanism by which the virus avoids clearance by the host is unknown. To determine if amino acid polymorphisms in the GB virus type C (GBV-C) NS5A and E2 proteins alter response to interferon (IFN) therapy, we studied the sequence of GBVC NS5A and E2 obtained from people receiving IFN therapy. In addition, we expressed recombinant GBVC NS5A protein to determine if it interferes with RNA-activated protein kinase (PKR) function in vitro. GBVC NS5A amplified from a person whose virus was cleared by IFN therapy (IFN sensitive) demonstrated unique amino acid changes occurring in the region that aligns with the hepatitis C virus (HCV) IFN sensitivity-determining region (ISDR) compared with NS5A sequences from individuals who did not clear GBV-C (IFN resistant). There were no differences in the E2 sequences obtained from IFN-sensitive and IFN-resistant isolates. Using a yeast genetic system, IFN-resistant NS5A inhibited PKR-mediated phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) in yeast, whereas IFN-sensitive NS5A did not inhibit PKR function. GBV-C NS5A amino acid polymorphisms appear to be involved in response to IFN therapy, and IFN-resistant GBV-C NS5A inhibited PKR-mediated eIF2alpha phosphorylation in a yeast genetic system, suggesting a mechanism by which GBV-C may evade clearance by naturally occurring host antiviral responses.

Amino Acid Sequence↗
PubMed2005

Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I.

Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.

Base Sequence↗
PubMed2004

Strategies for hepatitis C therapeutic intervention: now and next.

Interferon-alpha-based therapies are the mainstay of hepatitis C treatment. However, these broad immunomodulatory agents, already limited by their side effects, expense and route of administration, are not efficacious in all cases. Therefore, research efforts have focused on identifying additional drugs and therapeutic modalities to benefit those individuals refractive to current therapy, and which might be more affordable, such as orally active small-molecule alternatives and preventive or therapeutic vaccines. It is likely that successful therapeutic intervention of hepatitis C will require a combination of approaches targeting different aspects of the viral life-cycle.

Animals↗
PubMed2004

The host response to West Nile Virus infection limits viral spread through the activation of the interferon regulatory factor 3 pathway.

Recent outbreaks of West Nile Virus (WNV) have been associated with an increase in morbidity and mortality in humans, birds, and many other species. We have initiated studies to define the molecular mechanisms by which a recent pathogenic isolate of WNV evades the host cell innate antiviral response. Biochemical and microarray analyses demonstrated that WNV induced the expression of beta interferon (IFN-beta) and several IFN-stimulated genes late in infection of cultured human cells. The late expression of these antiviral genes was due to the delayed activation of the transcription factor IFN regulatory factor 3 (IRF-3). Despite this host response, WNV was still able to replicate efficiently. The effect of the IRF-3 pathway on WNV replication was assessed by examining virus replication and spread in cultures of wild-type or IRF-3-null mouse embryo fibroblasts. The absence of IRF-3 was marked by a significant increase in plaque size and a sustained production of infectious particles. Although the activation of the IRF-3 pathway was not sufficient to block virus replication, our results suggest that IRF-3 target genes function to constrain WNV infection and limit cell-to-cell virus spread.

Animals↗
PubMed2004

Viral evolution and interferon resistance of hepatitis C virus RNA replication in a cell culture model.

Hepatitis C virus (HCV) replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. We evaluated HCV-IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV type 1b subgenomic RNA replicon that differed at only two sites within the NS5A-encoding region. A replicon with a K insertion at HCV codon 2040 replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an L2198S point mutation replicated poorly and triggered a cellular response characterized by IFN-beta production and low-level IFN-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high-passage (HP) variant with six additional point mutations throughout the HCV protein-encoding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1,000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-alpha treatment and was associated with virus-directed reduction in host cell expression of ISG56, an antagonist of HCV RNA translation. Accordingly, the HP RNA was retained within polyribosome complexes in vivo that were refractory to IFN-induced disassembly. These results identify ISG56 as a translational control effector of the host response to HCV and provide direct evidence to link this response to viral sequence evolution, ISG regulation, and selection of the IFN-resistant viral phenotype.

Biological Evolution↗
PubMed2003

Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation.

Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.

Antiviral Agents↗
PubMed2003

Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease.

Persistent infections with hepatitis C virus (HCV) are likely to depend on viral inhibition of host defenses. We show that the HCV NS3/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3 phosphorylation after cellular challenge with an unrelated virus. Furthermore, dominant-negative or constitutively active IRF-3 mutants, respectively, enhanced or suppressed HCV RNA replication in hepatoma cells. Thus, the NS3/4A protease represents a dual therapeutic target, the inhibition of which may both block viral replication and restore IRF-3 control of HCV infection.

DNA-Binding Proteins↗
PubMed2003

The regulation of hepatitis C virus (HCV) internal ribosome-entry site-mediated translation by HCV replicons and nonstructural proteins.

Hepatitis C virus (HCV), the global leading cause of chronic liver disease, has a positive-sense, ssRNA genome that encodes a large polyprotein. HCV polyprotein translation is initiated by an internal ribosome-entry site (IRES) located at the 5' end of the viral genome, in a cap-independent manner, but the regulatory mechanism of this process remains poorly understood. In this study, we characterized the effect of HCV nonstructural proteins on HCV IRES-directed translation in both HCV replicon cells and transiently transfected human liver cells expressing HCV nonstructural proteins. Using bicistronic reporter gene constructs carrying either HCV or other viral IRES sequences, we found that the HCV IRES-mediated translation was specifically upregulated in HCV replicon cells. This enhancement of HCV IRES-mediated translation by the replicon cells was inhibited by treatment with either type I interferon or ribavirin, drugs that perturb HCV genome replication, suggesting that the enhancement is probably due to HCV-encoded protein function(s). Reduced phosphorylation levels of both eIF2alpha and eIF4E were observed in the replicon cells, which is consistent with our previous findings and indicates that the NS5A nonstructural protein may be involved in the regulatory mechanism(s). Indeed, transient expression of NS5A or NS4B in human liver cells stimulated HCV IRES activity. Interestingly, mutation in the ISDR of NS5A perturbed this stimulation of HCV IRES activity. All these results suggest, for the first time, that HCV nonstructural proteins preferentially stimulate the viral cap-independent, IRES-mediated translation.

Antiviral Agents↗
PubMed2003

Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication.

Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2alpha) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2alpha phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively, target eIF2- and eIF3-dependent steps in the viral RNA translation initiation process.

Adaptor Proteins, Signal Transducing↗
PubMed2002

P52rIPK regulates the molecular cochaperone P58IPK to mediate control of the RNA-dependent protein kinase in response to cytoplasmic stress.

The 52 kDa protein referred to as P52(rIPK) was first identified as a regulator of P58(IPK), a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52(rIPK) and P58(IPK) each possess structural domains implicated in stress signaling, including the charged domain of P52(rIPK) and the tetratricopeptide repeat (TPR) and DnaJ domains of P58(IPK). The P52(rIPK) charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52(rIPK) function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58(IPK). This domain bound specifically to P58(IPK) TPR domain 7, the domain adjacent to the TPR motif required for P58(IPK) interaction with PKR, thus providing a mechanism for P52(rIPK) inhibition of P58(IPK) function. Both the charged domain of P52(rIPK) and the TPR 7 domain of P58(IPK) were required for P52(rIPK) to mediate downstream control of PKR activity, eIF2alpha phosphorylation, and cell growth. Furthermore, we found that P52(rIPK) and P58(IPK) formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52(rIPK) charged domain functions as a TPR-specific signaling motif to directly regulate P58(IPK) within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.

3T3 Cells↗
PubMed2002

Regulation of PKR and IRF-1 during hepatitis C virus RNA replication.

The virus-host interactions that influence hepatitis C virus (HCV) replication are largely unknown but are thought to involve those that disrupt components of the innate intracellular antiviral response. Here we examined cellular antiviral pathways that are triggered during HCV RNA replication. We report that (i) RNA replication of HCV subgenomic replicons stimulated double-stranded RNA (dsRNA) signaling pathways within cultured human hepatoma cells, and (ii) viral RNA replication efficiency corresponded with an ability to block a key cellular antiviral effector pathway that is triggered by dsRNA and includes IFN regulatory factor-1 (IRF-1) and protein kinase R (PKR). The block to dsRNA signaling was mapped to the viral nonstructural 5A (NS5A) protein, which colocalized with PKR and suppressed the dsRNA activation of PKR during HCV RNA replication. NS5A alone was sufficient to block both the activation of IRF-1 and the induction of an IRF-1-dependent cellular promoter by dsRNA. Mutations that clustered in or adjacent to the PKR-binding domain of NS5A relieved the blockade to this IRF-1 regulatory pathway, resulting in induction of IRF-1-dependent antiviral effector genes and the concomitant reduction in HCV RNA replication efficiency. Our results provide further evidence to support a role for PKR in dsRNA signaling processes that activate IRF-1 during virus infection and suggest that NS5A may influence HCV persistence by blocking IRF-1 activation and disrupting a host antiviral pathway that plays a role in suppressing virus replication.

DNA-Binding Proteins↗
PubMed2002

Viruses and interferon: a fight for supremacy.

The action of interferons (IFNs) on virus-infected cells and surrounding tissues elicits an antiviral state that is characterized by the expression and antiviral activity of IFN-stimulated genes. In turn, viruses encode mechanisms to counteract the host response and support efficient viral replication, thereby minimizing the therapeutic antiviral power of IFNs. In this review, we discuss the interplay between the IFN system and four medically important and challenging viruses -- influenza, hepatitis C, herpes simplex and vaccinia -- to highlight the diversity of viral strategies. Understanding the complex network of cellular antiviral processes and virus-host interactions should aid in identifying new and common targets for the therapeutic intervention of virus infection. This effort must take advantage of the recent developments in functional genomics, bioinformatics and other emerging technologies.

Animals↗
PubMed2002

Activation of the interferon-beta promoter during hepatitis C virus RNA replication.

In this study we examined the impact of hepatitis C virus (HCV) RNA replication on the innate antiviral response of the host cell. Replication of an HCV subgenomic replicon stimulated the activation of the interferon (IFN)-beta promoter and the production of IFN in human hepatoma cells. Using a variety of functional assays, we found that HCV RNA replication induced the activation and DNA-binding activity of NFkappaB and interferon regulatory factor (IRF)-1. In addition, microscopy experiments revealed a higher frequency of cells containing the nuclear-localized, active form of IRF-3 in HCV replicon cultures versus control cultures. Consistent with these observations, cells harboring the HCV replicon exhibited high basal level expression of a subset of IFN-stimulated antiviral genes. Our results indicate that HCV RNA replication can stimulate cellular antiviral programs that contribute to the assembly and activation of the IFN-beta enhanceosome complex and initiation of the antiviral state. Stable HCV RNA replication in the face of the host antiviral response suggests that HCV may encode one or more proteins capable of overcoming specific antiviral processes, thereby supporting persistent infection.

2',5'-Oligoadenylate Synthetase↗
PubMed2002

Expression of human PKR protein kinase in transgenic mice.

There is a large amount of evidence describing the expression, interaction, and mode of activation of the human interferon (IFN)-mediated double-stranded RNA-activated protein kinase (PKR) gene. Studies from Pkr-null mice have defined the kinase as a transducer of dsRNA signals that converge on transcription, translation, and apoptotic programs involved in the innate resistance to viral infection. In vitro studies also suggest that PKR may possess important cell growth regulatory and tumor suppressor properties. However, the study of Pkr-null mice has not fully elucidated the role that the kinase plays in these processes, in part because of apparent redundancies in PKR-dependent and PKR-independent regulatory pathways. To overcome such limitations and to begin to examine the role of PKR in a complex biologic system, we have generated transgenic mice overexpressing wild-type human (Hu) PKR. HuPKR was expressed and active in various tissues and associated with a small body phenotype. Spleen cells from transgenic mice were resistant to apoptosis when treated with the genotoxic agent actinomycin D and showed a decrease in proliferation in response to concanavalin A (ConA) compared with spleen cells from wild-type control mice. The initial characterization of this transgenic mouse line suggests it may be useful as a model for investigating biology and diseases relative to a number of scientific disciplines.

Animals↗
PubMed2002

Effect of ankle taping and bracing on vertical ground reaction forces during drop landings before and after treadmill jogging.

STUDY DESIGN: Single-group repeated-measures experimental design. OBJECTIVES: The purpose of this study was to evaluate the effects of prophylactic ankle stabilization on vertical ground reaction forces before and after treadmill jogging. BACKGROUND: Previous research has demonstrated acute effects of ankle taping and bracing on ankle joint kinematics and vertical ground reaction forces during drop landings. Based on the number of investigations demonstrating increased range of motion of the braced or taped ankle following exercise, it may be plausible that the aforementioned landing alterations may return to normal following an exercise bout. METHODS AND MEASURES: Fourteen healthy recreational participants performed stiff and soft drop landings before and after a 20-minute treadmill exercise bout under 3 different ankle stabilizer conditions (no stabilizer, ankle brace, and ankle tape). A forceplate was used to collect ground reaction force data under the dominant foot. The first and second peak impact force, as well as the time to each of the 2 peak forces, were determined for each trial and used as dependent variables. RESULTS: The time to reach peak forces were significantly less under the ankle brace and tape conditions in comparison to the control (no-stabilizer) condition. CONCLUSIONS: It appears that ankle taping and bracing decrease the time to reach peak impact forces. These alterations indicate that during dynamic activity the musculoskeletal structures of the body may be subjected to loads within shorter time periods. Whether these effects are detrimental over time remains speculative at this point and requires further research.

Adaptation, Physiological↗