Perturbation of epidermal growth factor receptor complex formation and Ras signalling in cells harbouring the hepatitis C virus subgenomic replicon

Macdonald, Andrew; Chan, Julia Ka Yu; Harris, Mark

doi:10.1099/vir.0.80724-0

Cited by 20 publications

(19 citation statements)

References 30 publications

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“…Previous studies have demonstrated the ability of NS5A of genotype 1 HCV to inhibit signalling of the Ras-Erk pathway (Georgopoulou et al, 2003;Macdonald et al, 2003Macdonald et al, , 2005aTan et al, 1999); our data show that this ability is conserved by NS5A from the genotype 2a JFH-1 isolate, consistent with the absolute conservation of the class II polyproline motif (PP2) throughout all HCV isolates. Furthermore, this inhibition occurs within the first 24 h p.t., suggesting it is required in the early stages of the viral life cycle.…”

Section: Discussionsupporting

confidence: 87%

“…We have previously shown that NS5A of HCV genotype 1 inhibited Ras-Erk signalling, either when expressed alone or in the context of a culture-adapted subgenomic replicon or full-length HCV expression construct (Macdonald et al, 2003(Macdonald et al, , 2005aMankouri et al, 2008). To test the effects of the Ras-Erk pathway on HCV RNA replication we chose to use the recently developed genotype 2a-based transient luciferase replicon SGR-luc-JFH-1 (Targett- Adams & McLauchlan, 2005), because this replicon (unlike the genotype 1b versions) demonstrated high levels of replication within a short timescale (,24 h), which would allow quantitative measurement of viral RNA replication in the presence of inhibitors of Ras-Erk signalling.…”

Section: Perturbation Of Ras-erk Signalling Occurs In the Context Of mentioning

confidence: 99%

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Hepatitis C virus RNA replication is regulated by Ras-Erk signalling

Gretton

Hughes

Harris

2009

Journal of General Virology

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The hepatitis C virus NS5A protein has been previously demonstrated to partially attenuate activation of the Ras-Erk signalling pathway, via a conserved class II polyproline motif located towards the C terminus of the protein. However, the role of Ras-Erk signalling in the virus life cycle remains undetermined. To investigate this, levels of RNA replication were measured in genotypes 1 and 2 transient luciferase subgenomic replicon systems in the context of either pharmacological or genetic (dominant-negative) inhibition of MEK1, a kinase in the Ras-Erk signalling cascade. Incubation in the presence of two inhibitors (U0126 and PD184352) resulted in a decrease in the levels of RNA replication, conversely incubation with inhibitor PD98059 resulted in a modest increase in replication. The results obtained with PD98059 could not be explained by an off-target effect on Cox-2, stability of replicon RNA or stimulation of global translation levels, suggesting stimulation by a yet uncharacterized mechanism. To verify data obtained using pharmacological inhibitors the transient replicon RNA was co-electroporated with a dominant-negative mutant of MEK1. This resulted in a reduction in replication, confirming data seen with U0126 and PD184352. Our data are consistent with the hypothesis that a low level Ras-Erk signalling activity is required for RNA replication. However, complete inhibition of Ras-Erk signalling is inhibitory. These results suggest that perturbation of this signalling pathway by NS5A may be a mechanism to regulate levels of genomic RNA replication.

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Section: Discussionsupporting

confidence: 87%

Section: Perturbation Of Ras-erk Signalling Occurs In the Context Of mentioning

confidence: 99%

Hepatitis C virus RNA replication is regulated by Ras-Erk signalling

Gretton

Hughes

Harris

2009

Journal of General Virology

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“…Fewer studies have demonstrated a negative correlation between ERK activity and viral protein expression such as we have described. One such study demonstrated that the hepatitis C virus nonstructural NS5A protein inhibits EGF-stimulated activation of the ERK pathway by inhibiting EGFR complex formation (27). A recent study by Yoshizuka et al reported that the expression of the HIV type 1 (HIV-1) Vpr protein is associated with the downregulation of genes in the ERK/ MAPK pathway and with decreased phosphorylation of ERK1/2 (49).…”

Section: Discussionmentioning

confidence: 99%

The ERK Mitogen-Activated Protein Kinase Pathway Contributes to Ebola Virus Glycoprotein-Induced Cytotoxicity

Zampieri

Fortin

Nolan

et al. 2007

J Virol

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Ebola virus is a highly lethal pathogen that causes hemorrhagic fever in humans and nonhuman primates. Among the seven known viral gene products, the envelope glycoprotein (GP) alone induces cell rounding and detachment that ultimately leads to cell death. Cellular cytoxicity is not seen with comparable levels of expression of a mutant form of GP lacking a mucin-like domain (GP⌬muc). GP-induced cell death is nonapoptotic and is preceded by downmodulation of cell surface molecules involved in signaling pathways, including certain integrins and epidermal growth factor receptor. To investigate the mechanism of GP-induced cellular toxicity, we analyzed the activation of several signal transduction pathways involved in cell growth and survival. The active form of extracellular signal-regulated kinases types 1 and 2 (ERK1/2), phospho-ERK1/2, was reduced in cells expressing GP compared to those expressing GP⌬muc as determined by flow cytometry, in contrast to the case for several other signaling proteins. Subsequent analysis of the activation states and kinase activities of related kinases revealed a more pronounced effect on the ERK2 kinase isoform. Disruption of ERK2 activity by a dominant negative ERK or by small interfering RNA-mediated ERK2 knockdown potentiated the decrease in ␣V integrin expression associated with toxicity. Conversely, activation of the pathway through the expression of a constitutively active form of ERK2 significantly protected against this effect. These results indicate that the ERK signaling cascade mediates GP-mediated cytotoxicity and plays a role in pathogenicity induced by this gene product.Ebola virus is an enveloped, negative-strand RNA virus in the family Filoviridae, which is capable of inducing severe hemorrhagic fever syndrome in humans, nonhuman primates, and other species (37). Four species of the virus have been identified to date: Zaire, Sudan, Ivory Coast, and Reston. Of these, the highly pathogenic Zaire species produces mortality rates of up to 90% (23). Though the pathogenic determinants of Ebola virus remain incompletely defined, several lines of evidence suggest that the viral glycoprotein (GP) is a key contributor to the adverse events of infection. Expression of Ebola virus GP from all four subtypes induces variable degrees of cytotoxicity in cell lines and primary cells in vitro that is characterized by cell rounding and detachment, followed by cell death (47). Although there is some debate over the role of GP cytotoxicity during live viral infection (2, 18, 35), differences in GP-induced cytotoxicity are reflected in the mortality rates caused by the different subtypes (40, 47), indicating the importance of this gene product in the pathogenic course of the disease.Membrane-associated Ebola virus GP is a heavily glycosylated type I transmembrane protein that is responsible for receptor binding and fusion of the virus with host cells (9,37,46). During assembly, a GP precursor (GP0) is cleaved by a furinlike protease into GP1 and GP2 subunits to form a heterodimer. Trime...

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“…Previous work by our group (Macdonald et al, 2003(Macdonald et al, , 2005a and others (Georgopoulou et al, 2003) has shown that NS5A, when expressed either alone or in the context of the complete polyprotein, is able to block Ras-Erkpathway-mediated activation of the AP-1 transcription factor in a manner that was dependent on the presence of a conserved C-terminal polyproline motif (consensus sequence PxxPxR) within NS5A. As GBV-B NS5A lacks this PxxPxR motif we predicted that GBV-B NS5A would be unable to modulate this mitogenic signalling cascade.…”

Section: Gbv-b Ns5a Does Not Inhibit Activation Of the Ap-1 Transcripmentioning

confidence: 99%

“…In addition NS5A has been shown to bind to and modulate a range of cellular proteins, influencing activation of signalling pathways . Of particular interest is the observation that NS5A inhibits the Ras-Erk mitogen-activated protein kinase (MAPK) pathway acting between the epidermal growth factor receptor (EGFR) and the activation of Ras (Georgopoulou et al, 2003;Macdonald et al, 2003Macdonald et al, , 2005aTan et al, 1999). Furthermore, NS5A has also been shown to bind to, and activate, phosphoinositide 3-kinase (PI3K), resulting in activation of the downstream effector serine/ threonine kinase Akt/protein kinase B (He et al, 2002;Street et al, 2004) and elevating b-catenin-dependent transcription (Street et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

A comparative cell biological analysis reveals only limited functional homology between the NS5A proteins of hepatitis C virus and GB virus B

Mankouri

Milward

Pryde

et al. 2008

Journal of General Virology

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GB virus B (GBV-B) is the closest relative to hepatitis C virus (HCV) with which it shares a common genome organization, however, unlike HCV in humans, it generally causes an acute resolving hepatitis in New World monkeys. It is important to understand the factors regulating the different disease profiles of the two viruses and in this regard, as well as playing a key role in viral RNA replication, the HCV NS5A non-structural protein modulates a variety of host-cell signalling pathways. We have shown previously that HCV NS5A, expressed either alone, or in the context of the complete polyprotein, inhibits the Ras-extracellular-signal-regulated kinase (Erk) pathway and activates the phosphoinositide 3-kinase (PI3K) pathway. In this report, we investigate whether these functions are shared by GBV-B NS5A. Immunofluorescence analysis revealed that a C-terminally FLAG-tagged GBV-B NS5A exhibited a punctate cytoplasmic distribution. However, unlike HCV NS5A, the GBV-B protein did not partially co-localize with early endosomes. Utilizing a transient luciferase reporter system, we observed that GBV-B NS5A failed to inhibit Ras-Erk signalling, however GBV-B NS5A expression did result in the elevation of b-catenin-dependent transcription via activation of the PI3K pathway. These effects of GBV-B and HCV NS5A on the PI3K and Ras-Erk pathways were confirmed in cells harbouring subgenomic replicons derived from the two viruses. Based on these data we speculate that the differential effects of the two NS5A proteins on cellular signalling pathways may contribute to the differences in the natural history of the two viruses. INTRODUCTIONGB virus B (GBV-B) is the closest phylogenetic relative of hepatitis C virus (HCV) (Muerhoff et al., 1995;Ohba et al., 1996); it shares up to 28 % overall amino acid identity with HCV, rising to 50 % conservation in regions such as the NS3 coding sequence. GBV-B has been proposed as a model for HCV infection and has been used to characterize candidate antivirals as well as for pathogenesis studies (Beames et al., 2001;Bright et al., 2004). It shares the hepatotropism of HCV. Although GBV-B has been reported to lead to persistent infections in some cases in tamarins (Saguinus species) (Martin et al., 2003;Nam et al., 2004), unlike the human virus, GBV-B primarily causes an acute, resolving hepatitis in tamarins and other New World monkeys such as common marmosets (Callithrix jacchus) (Bukh et al., 2001;Jacob et al., 2004;Lanford et al., 2003). In contrast, HCV is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, affecting an estimated 3 % of the world's population.Both GBV-B and HCV are members of the genus Hepacivirus of the family Flaviviridae and have a positivesense RNA genome that is translated in a cap-independent fashion to generate a polyprotein, cleaved by host and viral proteases to produce 10 mature viral proteins. There are three structural proteins at the N terminus: core, E1 and E2, followed by a small cation channel, p7 (p13 in GBV-B). The C-terminal t...

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Perturbation of epidermal growth factor receptor complex formation and Ras signalling in cells harbouring the hepatitis C virus subgenomic replicon

Cited by 20 publications

References 30 publications

Hepatitis C virus RNA replication is regulated by Ras-Erk signalling

Hepatitis C virus RNA replication is regulated by Ras-Erk signalling

The ERK Mitogen-Activated Protein Kinase Pathway Contributes to Ebola Virus Glycoprotein-Induced Cytotoxicity

A comparative cell biological analysis reveals only limited functional homology between the NS5A proteins of hepatitis C virus and GB virus B

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