Mutational Analysis of Different Regions in the Coxsackievirus 2B Protein

Jong, Arjan S. de; Melchers, Willem J. G.; Glaudemans, Dirk H. R. F.; Willems, Peter H.G.M.; Kuppeveld, Frank J. M. van

doi:10.1074/jbc.m314094200

Cited by 41 publications

(12 citation statements)

References 57 publications

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“…Mutations in the 2B protein cause early defects in viral RNA replication (6,42,43). Moreover, our previous reports suggest that there is a close correlation between the ability of 2B mutants to alter membrane permeability and to support viral RNA replication (23,40). Infected cells contain both the 2B protein and its relatively stable precursor 2BC.…”

Section: Discussionmentioning

confidence: 94%

“…The mutants in question, ins (5)linker, ins (34)linker, and ins (94)linker mutants, contain a 9-amino acid linker inserted at the indicated amino acid position (23). All three mutants localized to the Golgi complex and inhibited VSV-G-GFP trafficking through this complex (Fig.…”

Section: The 2b Protein Disturbs Er and Golgi Ca 2ϩ Homeostasis Withomentioning

confidence: 99%

“…The mutants in question, K41L/K44L/K48L and I64S/V66S, have mutations in the hydrophobic regions HR1 and HR2 that are implicated in pore formation (23). BGM cells were transfected with expression plasmids of the fluorescent VSV-G-GFP fusion protein and mutant 2B-Myc constructs and processed as described above.…”

Section: The 2b Protein Disturbs Er and Golgi Ca 2ϩ Homeostasis Withomentioning

confidence: 99%

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The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

Jong¹,

Visch

Mattia³

et al. 2006

Journal of Biological Chemistry

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Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca 2؉ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca 2؉ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca 2؉ into the stores. Measurement of intra-organelle-free Ca 2؉ in permeabilized cells revealed that the ability of 2B to reduce the Ca 2؉ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cellfree system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca 2؉ -ATPase that transports Ca 2؉ from the cytosol into the stores. To investigate whether 2B specifically affects Ca 2؉ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H ؉ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.Enteroviruses (e.g. poliovirus, coxsackievirus, ECHOvirus) belong to the family of Picornaviridea, a large family of nonenveloped, cytolytic viruses that contain a single-stranded RNA genome of positive polarity. Upon infection, enteroviruses induce a number of dramatic alterations in their host cell, which serve to create the appropriate conditions for viral RNA replication and/or prevent antiviral host cell responses. One of these alterations is the modification of intracellular Ca 2ϩ homeostasis. We have previously shown that infection of HeLa cells with coxsackievirus results in a reduction of the amount of Ca 2ϩ that can be released from the intracellular stores using thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), 2 the Ca 2ϩ -ATPase that transports Ca 2ϩ from the cytosol into the stores. In addition, a gradual increase in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) was observed due to the influx of extracellular Ca 2ϩ (1). The enterovirus 2B protein, one of the nonstructural proteins involved in viral RNA replication, plays a major role in the alterations in intracellular Ca 2ϩ homeostasis that take place in enterovirus-infected cells (1, 2). The mechanism by which 2B, or its precursor 2BC, exerts its effects is largely unknown. Ca 2ϩ homeostasis in the intracellular stores (i.e. endoplasmic reticulum (ER) and Golgi) is the net result of the activity of the SERCA on the one hand and the continuous passive Ca 2ϩ leak from these organelles that exists under normal conditions on the other hand (3). Thus, the reductions in the Ca 2ϩ filling state of the stores ...

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Section: Discussionmentioning

confidence: 94%

Section: The 2b Protein Disturbs Er and Golgi Ca 2ϩ Homeostasis Withomentioning

confidence: 99%

Section: The 2b Protein Disturbs Er and Golgi Ca 2ϩ Homeostasis Withomentioning

confidence: 99%

See 1 more Smart Citation

The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

Jong¹,

Visch

Mattia³

et al. 2006

Journal of Biological Chemistry

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“…It has been shown for M2, however, that disulfide bonds between adjacent ectodomains help to stabilize the tetrameric complex (31). Asparagine residues located outside of the trans-membrane region have been implicated in oligomerization for Coxsackievirus 2B protein (34). There are two well conserved asparagine residues within the p7 sequence, both toward the N terminus in regions thought to lie outside of the trans-membrane domains (Asn-4 and Asn-9 in the 1b genotype), although these are not present in the genotype 2a JFH-1 sequence, which is able to undergo a complete replication cycle in culture (35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

Evidence for the Formation of a Heptameric Ion Channel Complex by the Hepatitis C Virus P7 Protein in Vitro

Clarke¹,

Griffin²,

Beales

et al. 2006

Journal of Biological Chemistry

119

136

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The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex. Hepatitis C virus (HCV)5 currently infects over 3% of the world's population and is the major indicator for liver transplantation in the developed world. Liver disease in the form of cirrhosis, liver failure, or hepatocellular carcinoma arises after many years of persistent virus infection following sub-clinical acute episodes. For this reason intervention is limited to chemotherapeutic treatment of chronic patients. The current regime of pegylated interferon ␣ and ribavirin, while effective against certain viral genotypes, is largely ineffective against the most common HCV genotype 1 (1, 2) viruses making the search for new HCV anti-viral drug targets crucial. A recent metaanalysis of multiple clinical trials where amantadine was included alongside current therapies showed that an improved sustained viral response was achieved in patients that had previously failed to respond to dual therapy (3).HCV is the prototype member of the Hepacivirus genus of the Flaviviridae family. It is an enveloped virus, and its genome comprises a single-stranded positive sense RNA of ϳ9.6 kb (4) that is translated from an internal ribosome entry site to yield a single polyprotein. This is cleaved by both host signalases and virus-encoded proteases yielding viral structural proteins and the non-structural proteins (5); the latter form the viral RNA replication complexes on modified cellular membranes and modulate host cell metabolism (6).The region of the HCV genome between the structural and non-structural regions encodes a 63-amino acid integral membrane protein known as p7 (7), comprising two trans-membrane domains separated by a short basic loop (7, 8). We identified the function of p7 as a cation channel whose activity in suspended lipid bilayers was sensitive to the drug amantadine, providing a potential mechanism for the proposed efficacy of this drug in HCV treatment (9). Others have subsequently identified alt...

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“…The cellular proteins of COPII have reportedly been used in the virus-induced production of vesicles [61]. 2B and the precursor 2BC contain two hydrophobic regions, which are α amphipathic a-helix domain, which is important in multimerization, integrating into the membrane of the host Golgi and ER complex, producing virus-induced vesicles, and forming the virporin complex [60,62-64]. The accumulation of 2B or 2BC proteins on Golgi changes the permeability of plasma membrane [56,62] and the disassembly of Golgi complex [65], causing cell lysis [57].…”

Section: Introductionmentioning

confidence: 99%

Viral and host proteins involved in picornavirus life cycle

et al. 2009

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Picornaviruses cause several diseases, not only in humans but also in various animal hosts. For instance, human enteroviruses can cause hand-foot-and-mouth disease, herpangina, myocarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, severe neurological complications, including brainstem encephalitis, meningitis and poliomyelitis, and even death. The interaction between the virus and the host is important for viral replication, virulence and pathogenicity. This article reviews studies of the functions of viral and host factors that are involved in the life cycle of picornavirus. The interactions of viral capsid proteins with host cell receptors is discussed first, and the mechanisms by which the viral and host cell factors are involved in viral replication, viral translation and the switch from translation to RNA replication are then addressed. Understanding how cellular proteins interact with viral RNA or viral proteins, as well as the roles of each in viral infection, will provide insights for the design of novel antiviral agents based on these interactions.

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Mutational Analysis of Different Regions in the Coxsackievirus 2B Protein

Cited by 41 publications

References 57 publications

The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

Evidence for the Formation of a Heptameric Ion Channel Complex by the Hepatitis C Virus P7 Protein in Vitro

Viral and host proteins involved in picornavirus life cycle

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