Ion Channels Formed by NB, an Influenza B Virus Protein

Sunstrom, N.A.; Premkumar, Louis S.; Premkumar, Anita; Ewart, Gary; Cox, G B; Gage, Peter W.

doi:10.1007/s002329900037

Cited by 79 publications

(79 citation statements)

References 14 publications

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“…The values we observed are slightly higher than the conductance levels of Յ50 pS observed for ion channels recorded in a 0.1 M electrolyte solution (19)(20)(21)(22). NB and Vpu, ion channel-forming proteins of influenza B virus and HIV-1, respectively, generate similar conductances of Ϸ50 pS and less (23)(24)(25). Membrane-spanning domains inserted into lipid bilayers also give rise to similar conductance levels (24,(26)(27)(28).…”

Section: N-7-oxanonyl-6-deoxy-dgjcontrasting

confidence: 53%

“…To block an artificialy reconstituted channel of either full-length NB protein of influenza B virus (25) or a channel made of peptides covering only the transmembrane domain of NB (38), the concentrations of amantadine needed are one order of magnitude higher, in the millimolar range. Amantadine is used as an antiviral drug against influenza A but not against influenza B virus.…”

Section: N-7-oxanonyl-6-deoxy-dgjmentioning

confidence: 99%

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The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

Pavlović

Neville²,

Argaud³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

333

302

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We show that hepatitis C virus (HCV) p7 protein forms ion channels in black lipid membranes. HCV p7 ion channels are inhibited by long-alkyl-chain iminosugar derivatives, which have antiviral activity against the HCV surrogate bovine viral diarrhea virus. HCV p7 presents a potential target for antiviral therapy.H epatitis C virus (HCV) is the major cause of chronic hepatitis with a significant risk of end-stage liver cirrhosis and hepatocellular carcinoma (1). HCV belongs to the family Flaviviridae, which consists of three genera: flaviviruses, pestiviruses, and hepaciviruses. In the absence of both a suitable small animal model and a reliable in vitro infectivity assay for HCV, potential antiviral drugs initially have been tested by using a related pestivirus, bovine viral diarrhea virus (BVDV) (2). BVDV in vitro infectivity assays were used to demonstrate that long-alkyl-chain iminosugar derivatives containing either the glucose analogue deoxynojirimycin (DNJ) or the galactose analogue deoxygalactonojirimycin (DGJ) are potent antiviral inhibitors (3).DNJ derivatives inhibit endoplasmic reticulum (ER) ␣-glucosidases I and II (4, 5), and this inhibition leads to the misfolding of many host-and virus-encoded glycoproteins, including the envelope glycoproteins of BVDV (6) and HCV (7). Previous experiments have shown that the antiviral effect of the longalkyl-chain derivative N-nonyl-DNJ (NN-DNJ) is more pronounced than that of the short-alkyl-chain derivative N-butyl-DNJ (NB-DNJ), although the latter achieves a more effective ER ␣-glucosidase inhibition in cellulo. In addition, long-alkylchain DGJ derivatives that are not recognized by and do not inhibit ER ␣-glucosidases also show potent antiviral activity (3). Therefore, ER ␣-glucosidase inhibition does not correlate directly with the observed antiviral effect and is ruled out as the sole antiviral mechanism.The additional mechanism of action apparently is associated with the length of the alkyl side chain, because the short-chain N-butyl-DGJ (NB-DGJ) shows no antiviral activity, whereas the long-alkyl-chain derivative NN-DGJ is a potent inhibitor (3).The predominant antiviral mechanism is proposed to be mediated directly or indirectly by an effect of the long-alkyl side chains on the membrane and͞or membrane proteins, because treatment with long-alkyl-chain iminosugars affects the dimerization of viral membrane glycoproteins and alters the membrane glycoprotein composition of secreted BVDV virions but does not influence either viral RNA replication or protein synthesis (3).We decided to investigate the small membrane-spanning protein p7 as a potential target of long-alkyl-chain iminosugar derivatives, because flaviviruses such as dengue virus and Japanese encephalitis virus (8), which do not contain p7, are not inhibited by long-alkyl-chain DGJ derivatives, whereas pestiviruses are (3). Pesti-and hepaciviruses both contain the p7 protein.Most functional data about p7 are derived from the pestivirus p7, a 70-aa protein very similar to HCV p7. Functional data hav...

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Section: N-7-oxanonyl-6-deoxy-dgjcontrasting

confidence: 53%

Section: N-7-oxanonyl-6-deoxy-dgjmentioning

confidence: 99%

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

Pavlović

Neville²,

Argaud³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

333

302

View full text Add to dashboard Cite

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“…Levels of 20, 61, and 107 pS from experiments perfectly merge with the computational values. The higher conductance levels (251 and 413 pS) might be the consequence of assemblies greater than six segments with undefined topology and multiple channel openings due to the peptide concentration (7).…”

Section: Sequencementioning

confidence: 99%

“…However, when NB is incorporated in liposomes and then added to planar lipid bilayers, the conductance ranged from ca. 10 pS at very low NB concentration in the liposomes to several hundred picosiemens for higher concentrations (7). At neutral pH the channels are more permeable for sodium ions than chloride ions (P Na /P Cl ≈ 9 at pH 6.5).…”

mentioning

confidence: 97%

Transmembrane Peptide NB of Influenza B: A Simulation, Structure, and Conductance Study

et al. 2000

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The putative transmembrane segment of the ion channel forming peptide NB from influenza B was synthesized by standard solid-phase peptide synthesis. Insertion into the planar lipid bilayer revealed ion channel activity with conductance levels of 20, 61, 107, and 142 pS in a 0.5 M KCl buffer solution. In addition, levels at -100 mV show conductances of 251 and 413 pS. A linear current-voltage relation reveals a voltage-independent channel formation. In methanol and in vesicles the peptide appears to adopt an R-helical-like structure. Computational models of R-helix bundles using N ) 4, 5, and 6 NB peptides per bundle revealed water-filled pores after 1 ns of MD simulation in a solvated lipid bilayer. Calculated conductance values [using HOLE (Smart et al. (1997) Biophys. J. 72, 1109-1126] of ca. 20, 60, and 90 pS, respectively, suggested that the multiple conductance levels seen experimentally must correspond to different degrees of oligomerization of the peptide to form channels.In the genome of influenza B, RNA segment 6 encodes two integral membrane proteins, NB and NA. NB is a protein of ca. 100 amino acids. It has ca. 18 extramembraneous residues on its N-terminal end, a 22-residue membrane spanning segment, and a ca. 60 residue long C-terminal end (1, 2). NB is expressed at the surface of infected cells (3)(4)(5). In the virion the N-terminal site is located at the external surface. Experiments with gold immunolabeled freezefractured virions indicate that the C-terminus is located at the inside of the virion (4). By analogy with the M2 protein of influenza A, it was suggested (reviewed in ref 6) that NB might be a channel-forming protein.Conductance measurements with purified NB expressed in Escherichia coli and reconstituted in lipid bilayers reveal a conductance of 45 pS. However, when NB is incorporated in liposomes and then added to planar lipid bilayers, the conductance ranged from ca. 10 pS at very low NB concentration in the liposomes to several hundred picosiemens for higher concentrations (7). At neutral pH the channels are more permeable for sodium ions than chloride ions (P Na /P Cl ≈ 9 at pH 6.5). In asymmetrical NaCl solutions at pH 2.5 the permeability is higher for chloride ions (P Cl / P Na ≈ 4). A recent study on NB expressed in the plasmalemma of MEL 1 cells showed that the channel peptides induce a Na + -dependent proton current and a pH-activated Cl -current (8).One way in which single integral membrane proteins can form ion channels is via oligomerization to form a bundle of transmembrane helices. For the analogous channel peptide in influenza A, M2, it is believed that a tetrameric R-helix bundle is the minimal form of the proton-permeable ion channel (9-11). For NB the number of subunits in the bundle is not known. Purification of NB on gels using nonreducing conditions revealed the formation of disulfide-linked dimers and higher oligomers (2). Molecular modeling studies on bundles of the transmembrane parts of the peptide have been performed in vacuo with four, five, and si...

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“…RNA segment 6 of influenza A virus is monocistronic, coding for the NA protein, whereas segment 6 of influenza B virus encodes NA and NB proteins by using a bicistronic mRNA with two overlapping ORFs. The NB protein of influenza B virus is a membrane protein that is believed to serve a function similar to that of the M2 protein of influenza A viruses (24)(25)(26). Furthermore, although RNA segment 7 of both influenza A and B viruses encodes the matrix protein M1, the organizations of their respective M2 genes are quite different.…”

mentioning

confidence: 99%

Rescue of influenza B virus from eight plasmids

Hoffmann

Mahmood

Yang

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

149

123

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Influenza B virus causes a significant amount of morbidity and mortality, yet the systems to produce high yield inactivated vaccines for these viruses have lagged behind the development of those for influenza A virus. We have established a plasmid-only reverse genetics system for the generation of recombinant influenza B virus that facilitates the generation of vaccine viruses without the need for time consuming coinfection and selection procedures currently required to produce reassortants. We cloned the eight viral cDNAs of influenza B͞Yamanashi͞166͞98, which yields relatively high titers in embryonated chicken eggs, between RNA polymerase I and RNA polymerase II transcription units. Virus was detected as early as 3 days after transfection of cocultured COS7 and Madin-Darby canine kidney cells and achieved levels of 10 6 -10 7 plaque-forming units per ml of cell supernatant 6 days after transfection. The full-length sequence of the recombinant virus after passage into embryonated chicken eggs was identical to that of the input plasmids. To improve the utility of the eight-plasmid system for generating 6 ؉ 2 reassortants from recently circulating influenza B strains, we optimized the reverse transcriptase-PCR for cloning of the hemagglutinin (HA) and neuraminidase (NA) segments. The six internal genes of B͞Yamanashi͞166͞98 were used as the backbone to generate 6 ؉ 2 reassortants including the HA and NA gene segments from B͞Victoria͞504͞2000, B͞Hong Kong͞ 330͞2001, and B͞Hawaii͞10͞2001. Our results demonstrate that the eight-plasmid system can be used for the generation of high yields of influenza B virus vaccines expressing current HA and NA glycoproteins from either of the two lineages of influenza B virus.

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Ion Channels Formed by NB, an Influenza B Virus Protein

Cited by 79 publications

References 14 publications

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

Transmembrane Peptide NB of Influenza B: A Simulation, Structure, and Conductance Study

Rescue of influenza B virus from eight plasmids

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