1 Astbury Centre of Molecular Biology, School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
2 School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK
3 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
Correspondence
David J. Rowlands
D.J.Rowlands{at}bmb.leeds.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Published ahead of print on 14 November 2003 as DOI 10.1099/vir.0.19634-0.
Present address: PHLS Central Public Health Laboratory, Colindale, London NW9 5HT, UK.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HCV is the prototype member of the Hepacivirus genus of the family Flaviviridae (Robertson et al., 1998). The virus is enveloped and has a single-stranded positive-sense RNA genome of around 9·6 kb, which is replicated in the cytosol via a negative-strand intermediate. An internal ribosome entry site (IRES) drives translation of a single polyprotein of approximately 3000 amino acids which is then proteolytically cleaved by cellular and viral proteases into the mature virus gene products: Core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (Clarke, 1997
). Core, with the two viral glycoproteins E1 and E2, comprise the structural proteins of the virion. The non-structural (NS) proteins are thought to form a ribonucleoprotein complex with the virus genome that associates with intracellular membranes and is the site of RNA replication effected by the NS5B protein (Schmidt-Mende et al., 2001
; Egger et al., 2002
).
The p7 protein of HCV lies at the junction between the structural and non-structural regions of the virus polyprotein (Lin et al., 1994; Mizushima et al., 1994
), though it is not known whether p7 is a virion component. The protein is small (63 amino acids) and highly hydrophobic in nature, largely comprising two predicted trans-membrane alpha helices, and has been shown to be an integral membrane protein (Carrere-Kremer et al., 2002
). We previously identified the function for p7 as an oligomeric ion channel capable of mediating cation flow across artificial membranes, using p7 from the 1b genotype of HCV (Griffin et al., 2003
). Others have since demonstrated similar activity for a genotype 1a p7, suggesting a likely conservation of function across all HCV genotypes (Pavlovic et al., 2003
), though extended studies with p7s from various genotypes will be necessary to confirm this. This finding confirms that HCV p7 belongs to an expanding family of viral proteins known as viroporins, which are small hydrophobic proteins encoded by a variety of RNA viruses that oligomerize to form cation channels most often involved in virus assembly or entry/exit, though many have additional functions (Liljestrom et al., 1991
; Duff & Ashley, 1992
; Pinto et al., 1992
; Aldabe & Carrasco, 1995
; Loewy et al., 1995
; Tian et al., 1995
; Ewart et al., 1996
; Newton et al., 1997
; van Kuppeveld et al., 1997
; Gonzalez & Carrasco, 1998
). The best-characterized viroporin is the M2 ion-channel of influenza A virus: the target of the antiviral drug, amantadine (Hay et al., 1985
). We showed that p7 ion channel activity could also be inhibited by amantadine (Griffin et al., 2003
), present at a concentration shown to specifically inhibit M2 in vitro (Duff & Ashley, 1992
). Given that the p7 protein of the related pestivirus, bovine viral diarrhoea virus (BVDV), has been shown to be essential for the production of infectious virus particles (Harada et al., 2000
), it is likely that HCV p7 also plays an important role in the virus life-cycle.
As p7 is dispensable for RNA replication in subgenomic HCV replicons, it was necessary to use an alternative system to measure p7 ion channel activity in intact cells. Given that amantadine inhibits both M2 and p7 in vitro, we decided to test p7 in a cell-based assay designed to measure M2 function. We found that p7 was indeed functional in this assay and that its activity was inhibited by amantadine. In addition, the p7 of BVDV was also able to substitute for M2 in this assay, indicative of ion channel activity. As we have previously shown for HCV p7 (Griffin et al., 2003), the BVDV p7 protein was able to oligomerize both in vitro and in cells, further strengthening the case for these proteins being functional and structural homologues.
Mutation of the conserved cytosolic loop located between the trans-membrane alpha helices of HCV p7 abrogated ion channel function in this system. Co-expression of this mutant with wild-type HCV p7 inhibited wild-type activity. No defect in expression was caused by the mutation and both proteins displayed a similar intracellular localization. Co-localization of p7 with fluorescent markers for cellular organelles indicated that it largely accumulated in membranes associated with mitochondria. This work is the first demonstration of p7 ion channel function in living cells and provides a method for analysing the function of p7 mutants or screening potential inhibitory compounds.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mammalian cell culture and transfection.
Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10 % fetal calf serum, 100 IU ml-1 penicillin and 100 µg ml-1 streptomycin. For Vero cell transfection, cells were grown to 4070 % confluency, infected with a recombinant fowlpox virus (FPV) expressing T7 RNA polymerase for 1 h, and then transfected using Lipofectamine (Invitrogen), according to the manufacturer's instructions, with 1 µg HA cDNA and 0·2 µg M2/p7 cDNA per well of a six-well plate. 293T cells were grown to 6080 % confluency either in wells of a six-well plate or on coverslips coated in poly(L-lysine) (Sigma) in a 12-well plate well and transfected using Lipofectamine with 2 or 1 µg DNA respectively. Co-transfections of GFP fusion plasmids and CFP marker constructs were made up of 0·5 µg of each plasmid.
Haemadsorption assay.
This method is adapted from a published method (Takeuchi & Lamb, 1994; Medeiros et al., 2001
). Vero cells were infected and transfected as described above. 48 h post-transfection the cells were washed twice with PBS. 1 ml of 5·5 mU ml-1 bacterial neuraminidase from Vibrio cholerae (Roche) diluted in serum-free DMEM was added to each well. The cells were incubated at 37 °C for 1 h and then washed three times to remove any traces of neuraminidase before addition of 1 ml of a 0·5 % (w/v) suspension of horse red blood cells in PBS to each well. After incubation at room temperature for 2 h plates were gently shaken to resuspend unbound red blood cells and washed three times with PBS. Cells were lysed in 1 ml of CAT ELISA lysis buffer (Roche) for 3 min and the lysate was clarified in a microfuge at 13 000 r.p.m. for 5 min. Absorbance at 540 nm was recorded using a UV-2 UV/Vis spectrometer (Unicam).
Protein expression and immunoblotting.
For detection of expressed proteins, transfected 293T or Vero cells from one well of a six-well plate were washed twice and scraped into 1 ml PBS. Cells were pelleted at 7000 r.p.m. for 2 min and lysed in 100 µl of EBC lysis buffer (50 mM Tris/HCl pH 8·0, 140 mM NaCl, 100 mM NaF, 200 µM Na3VO4, 0·1 % SDS, 0·5 % NP40). Following normalization of protein concentration using the BCA protein assay (Bio-Rad), 10 µl of lysate was subjected to SDS-PAGE and transferred to an Immobilon PVDF membrane (Millipore). FLAG tagged proteins were detected with an anti-FLAG M2 mouse monoclonal antibody (Sigma) plus HRPgoat anti-mouse secondary (Sigma). BVDV p7 was detected using bovine hyperimmune anti-BVDV serum (donated by John McCauley, IAH, Compton, UK) plus HRPrabbit antibovine secondary (Sigma). H5 HA was detected using a polyclonal sheep antiserum plus HRPdonkey anti-sheep secondary (Sigma).
Radiolabelled BVDV p7 and HIS6-p7 were expressed in vitro using the TNT Quick coupled transcription/translation kit (Promega) with 1 µg of DNA and 10 µCi -Trans35S-label (ICN), according to manufacturers' instructions. Following RNase A treatment, proteins were precipitated with 2 vols of acetone and resuspended in 20 µl Laemmli buffer; the sample was then split and subjected to SDS-PAGE. Gels were immunoblotted as described or fixed in 40 % methanol/10 % acetic acid, and then treated with Enlightning (NEN-Dupont) for 30 min prior to autoradiography.
Fluorescence analysis of FLAGp7 and GFP fusions.
Cells to be labelled with MitoTracker red 580 nm (Molecular Probes) were incubated in a 200 nM solution of the dye in DMEM for 1 h prior to fixation. Cells transfected on coverslips were washed three times in PBS, fixed with 4 % paraformaldehyde in PBS for 20 min at room temperature and then washed twice more in PBS. GFP/CFP-labelled cells were analysed at this point. Cells to be analysed by immunocytochemistry were permeabilized with 1 % Triton X-100 in PBS or cold acetone (MitoTracker-labelled cells) for 5 min. Cells were washed three times and then incubated with FITC-conjugated mouse anti-FLAG monoclonal antibody (Sigma) diluted in 10 % fetal calf serum/PBS for 1 h at room temperature in a dark humidified container. Nuclei were labelled with Hoescht stain (Molecular Probes) diluted 1/10 000 in PBS and the cells washed a further three times in PBS and then once in distilled water prior to analysis. Images were captured using a DeltaVision restoration system (Applied Precision Inc.), based around an Olympus IX-70 inverted microscope. Optical sections of 0·2 µm were captured with a CoolSNAP HQ CCD camera (Roper Scientific). Digital deconvolution and image analysis were then performed on 3D datasets using 15 iterations of a constrained iterative deconvolution algorithm with SoftWoRx deconvolution software (Applied Precision Inc.).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
We previously showed that amantadine inhibited HCV p7 ion channel activity in artificial membranes (Griffin et al., 2003) and it was of interest to determine the effect of the drug in a cell-based assay. Amantadine (5 µM) reduced both M2- and p7-dependent haemadsorption to background levels observed when HA was transfected alone (Fig. 2c
). As inhibition of M2 at this concentration in cell culture is known to be specific, it would appear that amantadine also specifically inhibits HCV p7 ion channel activity.
The HCV p7 basic loop is essential for ion channel activity
HCV HIS6-p7 containing the KR mutation was assessed in the haemadsorption assay for ion channel activity. The mutation reduced the level of erythrocyte binding to baseline levels observed for HA alone (Fig. 2d). Furthermore, co-transfection (replacing half of the wild-type DNA with that of the mutant) of the KR mutant with wild-type p7 resulted in inhibition of the activity of the wild-type protein (Fig. 2d
). This implied that both the KR and wild-type p7 were present in the same intracellular compartment and formed hetero-oligomers incapable of functioning as ion channels. The number of functional ion channels required for a positive read-out in this assay cannot be determined: it is possible, therefore, that the presence of a small number of functional channels amongst an excess of hetero-oligomers can yield a measurable effect. In contrast, mutations of residues in other parts of the protein (V13L, C27A and P49A; data not shown) resulted in only a slight reduction in activity in the assay.
BVDV p7 forms an oligomeric ion channel
The amino acid sequences of the p7 proteins from BVDV and HCV are relatively distant (Fig. 1a), yet they share a high degree of predicted structural homology. It was of interest, therefore, to determine whether BVDV p7 could also function as an ion channel. A major feature of the viroporins is their ability to oligomerize in cellular membranes, as we have previously shown for HCV p7 (Griffin et al., 2003
). 35S-labelled BVDV p7 and BVDV HIS6-p7 were generated by in vitro translation in the presence of microsomal membranes and analysed by reducing SDS-PAGE. Both monomer and a higher molecular mass form, consistent with an oligomeric form of p7, were evident (Fig. 3
a). Both the native p7 and the HIS-p7 oligomers were estimated to be pentameric by comparison with molecular mass standards. Interestingly, these complexes were extremely stable compared to HCV p7 oligomers, which required stabilization with chemical cross-linkers (Griffin et al., 2003
). Furthermore, immunoblotting the same in vitro trans<1?show=[fo]>lation reactions using bovine hyperimmune anti-BVDV serum detected only the oligomeric form, providing circumstantial evidence that this is the predominant form in which BVDV p7 exists in natural infection. This form was also the only species detected when BVDV p7 was expressed in mammalian cells (Fig. 3a
).
|
p7 partially localizes to an intracellular compartment associated with mitochondria
The negative effect on wild-type p7 function observed for the KR mutant implied that both it and the wild-type localize to the same intracellular compartment. Others have previously observed a reticular distribution of HCV p7 expressed in human HepG2 hepatoma cells, though the cellular compartment that this represented was not identified (Carrere-Kremer et al., 2002). To analyse the subcellular distribution of p7 in more detail we generated expression constructs in which p7 was fused to eGFP at its C terminus and analysed its localization in transfected human 293T cells. These cells were found to express p7-GFP and other p7 derivatives most efficiently in a short time period, thereby avoiding cytotoxicity associated with long-term expression of these proteins. Similar fluorescence patterns were, however, observed in HepG2 and COS-7 cells transfected with these constructs (data not shown).
Surprisingly, expression of p7-GFP resulted in a punctate staining in the cytoplasm of transfected cells (Fig. 4b, c). This distribution was unaffected by the KR mutation and levels of expression appeared similar (Fig. 4d
). To determine the nature of the cellular compartment that this punctate staining represented, cells were co-transfected with p7-GFP and cyan fluorescent protein (CFP)-tagged markers for Golgi, endoplasmic reticulum (ER) and mitochondria (Living Colours, Clontech). Neither the ER nor the Golgi CFP fluorescence co-localized with p7-GFP making it unlikely that the punctate staining pattern had arisen due to disruption of these organelles (Fig. 4e, f
). The mitochondrial CFP marker, however, clearly overlapped with the signal from p7-GFP (Fig. 4g
). Furthermore, expression of BVDV p7 fused to eGFP resulted in a fluorescence pattern similar to that seen with HCV p7-GFP, and this also overlapped with the signal from the mitochondrial marker (Fig. 4h
).
It was important to ensure that the pattern of p7-GFP localization we observed was not an artefact of fusion to eGFP. We therefore expressed FLAG-p7 and stained permeabilized 293T cells with an FITC-conjugated monoclonal anti-FLAG antibody (Sigma) (Fig. 5a). Reassuringly, both FLAG-p7 and FLAG-p7KR displayed the same punctate form of staining as did the p7-GFP fusions (Fig. 5a
, panels 1 and 2), though a second pattern of a more reticular appearance was also evident (Fig. 5a
, panels 3 and 4). This pattern of staining was also observed when p7 with a C-terminal FLAG tag was expressed in the context of the other HCV structural proteins (Fig. 5c
).
To determine whether the reticular pattern of staining was also due to association with mitochondria, 293T cells expressing FLAG-p7 or p7-FLAG in the context of other HCV structural proteins were incubated with a mitochondrion-specific dye, MitoTracker red 580 nm (Molecular Probes). This dye specifically accumulates in the membranes of mitochondria in live cells and remains stable post-fixation. In all cases a significant overlap of the anti-FLAG FITC and MitoTracker signal was observed in cells positive for p7 expression, though regions positive for one or the other dye were also evident (Fig. 5b, d). This suggests that p7 was present not only in a mitochondrion-associated compartment, but also in adjacent membrane structures (Fig. 5b, d
).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amantadine abrogated the activity of p7 in the haemadsorption assay at concentrations known to specifically inhibit M2 in cell culture. This is consistent with our finding that the drug also inhibited channel formation in artificial membranes at similar concentrations (Griffin et al., 2003). Amantadine inhibits M2 by preventing the protonation of a histidine residue and its subsequent interaction with a tryptophan residue in the channel lumen, thus preventing channel opening (Duff et al., 1994
; Wang et al., 1995
; Salom et al., 2000
; Okada et al., 2001
). Interestingly, a conserved histidine residue is also present at position 17 in both HCV and BVDV p7 located within the N-terminal predicted trans-membrane alpha helix, though its role in ion channel function has yet to be determined. Amantadine has been used in clinical trials alongside current regimes with some success (Brillanti et al., 2000
; Cozzolongo et al., 2002
; Zilly et al., 2002
; Berg et al., 2003
; Piai et al., 2003
; Thuluvath et al., 2003
; Weegink et al., 2003
). The mode of action of amantadine in these cases is unclear, though we propose that it acts by inhibiting p7 ion channel function (Martin et al., 1999
; Jubin et al., 2000
; Griffin et al., 2003
).
The high degree of conservation in the basic loop region of HCV p7 suggests its importance for function and this was demonstrated by our finding that the KR mutant protein had lost ion channel activity. Our previous model of a p7 oligomeric channel predicted that the side-chains from K and R project into the channel lumen, perhaps forming a gate controlling the flow of ions (Griffin et al., 2003). Mutation of the analogous region in BVDV has previously been shown to abrogate virus infectivity in culture (Harada et al., 2000
). It is unlikely that the KR mutation caused decreased stability of the ion channel complex as co-expression with the wild-type protein resulted in a negative effect on wild-type function. The most plausible explanation for this is that the two proteins were present in the same intracellular compartment and were able to hetero-oligomerize forming a non-functional channel. In addition, it is likely that residues present in the trans-membrane alpha helices are responsible for the stable formation of oligomers, as has been suggested previously (Carrere-Kremer et al., 2002
).
During the preparation of this manuscript, it was shown that both deletion of p7 and mutating the KR loop abrogated replication of genotype 1a infectious RNA in chimpanzees (Sakai et al., 2003). Our demonstration that the KR loop is essential for genotype 1b p7 ion channel function makes it likely that this phenotype arose due to a similar defect in this activity for the 1a genotype. Furthermore, chimeras in which the 1a sequence was replaced with the 2a genotype were non-functional except in one case where the termini of the protein remained the parental genotype. This implied that p7 might interact with other viral gene products in a genotype-dependent fashion via these termini. Nevertheless, the demonstration that the 2a trans-membrane domains and KR loop could replace that of 1a supports the suggestion that p7 acts as an ion channel in all HCV genotypes.
The localization of p7 was unaffected by the KR mutation in transfected 293T cells. p7-GFP showed a punctate staining pattern, quite dissimilar to that shown in previous investigations of p7 cellular distribution (Carrere-Kremer et al., 2002) and this corresponded to at least partial localization to mitochondria. Expression of FLAG-p7 also gave a similar pattern in cells, though other more reticular staining was also apparent which significantly co-localized with mitochondria visualized with a specific stain. This pattern was also observed when p7 was expressed in the context of the other HCV structural proteins. Previously, p7 has been shown to display a reticular staining pattern in HepG2 cells leading to the supposition that the protein is mainly present in the ER, though no markers for cellular organelles were used to confirm this (Carrere-Kremer et al., 2002
). Interestingly, other HCV gene products have been shown to localize to membrane cisternae closely associated with mitochondria in replicon-bearing cells (Mottola et al., 2002
). An explanation for these different staining patterns could be that in dividing cells the morphology of mitochondria is known to vary between a classical punctate ovoid shape, with which they are usually associated, and a reticulum where the organelle is a continuous entity.
Recently, it has become apparent that a number of virally expressed proteins have the ability to localize to mitochondria and affect apoptotic pathways via alteration of mitochondrial membrane permeability: e.g. human immunodeficiency virus (HIV) Vpr (Jacotot et al., 2001), human T-cell lymphotropic virus (HTLV-1) p13II (D'Agostino et al., 2002
), hepatitis B virus (HBV) X protein (Takada et al., 1999
; Rahmani et al., 2000
) and influenza virus PB1 ORF 2 (Gibbs et al., 2003
). These are targeted to mitochondria via mechanisms differing from that of most cellular proteins, which possess canonical N-terminal targeting sequences. Instead, targeting signals are contained within the protein itself and are not proteolytically cleaved. The insertion of these proteins into mitochondrial membranes is thought to occur via a similar mechanism to that used by the cellular voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT) and Bcl-2 family proteins. These are major regulators of mitochondrial permeability (Halestrap & Brennerb, 2003
) and are known to interact with several of these viral proteins (Boya et al., 2001
, 2003
). It is likely that both HCV and BVDV p7 are targeted to mitochondrial membranes in a similar fashion, suggesting they may modulate apoptosis. This does not preclude that p7 possess other functions when present in other cellular compartments, as suggested by the observed incomplete overlap with mitochondrial markers. This is also the case for many other viroporins including HIV-1 Vpr, which is known to affect apoptosis when present in mitochondria yet plays several other roles in the virus life-cycle (Piller et al., 1996
, 1999
; Lamb & Pinto, 1997
; Halestrap & Brennerb, 2003
). For example, some data are suggestive of a role for p7 in the entry of HCV virus-like particles (Saunier et al., 2003
). Currently, the precise role of p7 in the HCV life-cycle is difficult to determine; however, given the importance of other viroporins for virus replication, the specific inhibition of HCV p7 by amantadine represents an opportunity for a new direction of research for antiviral chemotherapy.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berg, T., Kronenberger, B., Hinrichsen, H. & 10 other authors (2003). Triple therapy with amantadine in treatment-naive patients with chronic hepatitis C: a placebo-controlled trial. Hepatology 37, 13591367.[CrossRef][Medline]
Boya, P., Roques, B. & Kroemer, G. (2001). New EMBO members' review: viral and bacterial proteins regulating apoptosis at the mitochondrial level. EMBO J 20, 43254331.
Boya, P., Roumier, T., Andreau, K., Gonzalez-Polo, R. A., Zamzami, N., Castedo, M. & Kroemer, G. (2003). Mitochondrion-targeted apoptosis regulators of viral origin. Biochem Biophys Res Commun 304, 575581.[CrossRef][Medline]
Brillanti, S., Levantesi, F., Masi, L., Foli, M. & Bolondi, L. (2000). Triple antiviral therapy as a new option for patients with interferon nonresponsive chronic hepatitis C. Hepatology 32, 630634.[Medline]
Carrere-Kremer, S., Montpellier-Pala, C., Cocquerel, L., Wychowski, C., Penin, F. & Dubuisson, J. (2002). Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol 76, 37203730.
Choo, Q. L., Kuo, G., Weiner, A., Wang, K. S., Overby, L., Bradley, D. & Houghton, M. (1992). Identification of the major, parenteral non-A, non-B hepatitis agent (hepatitis C virus) using a recombinant cDNA approach. Semin Liver Dis 12, 279288.[Medline]
Ciampor, F., Bayley, P. M., Nermut, M. V., Hirst, E. M., Sugrue, R. J. & Hay, A. J. (1992). Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans Golgi compartment. Virology 188, 1424.[Medline]
Clarke, B. (1997). Molecular virology of hepatitis C virus. J Gen Virol 78, 23972410.
Cozzolongo, R., Cuppone, R. & Manghisi, O. G. (2002). The treatment of chronic hepatitis C not responding to interferon. Curr Pharm Des 8, 967975.[Medline]
D'Agostino, D. M., Ranzato, L., Arrigoni, G. & 10 other authors (2002). Mitochondrial alterations induced by the p13II protein of human T-cell leukemia virus type 1. Critical role of arginine residues. J Biol Chem 277, 3442434433.
Duff, K. C. & Ashley, R. H. (1992). The transmembrane domain of influenza A M2 protein forms amantadine-sensitive proton channels in planar lipid bilayers. Virology 190, 485489.[Medline]
Duff, K. C., Gilchrist, P. J., Saxena, A. M. & Bradshaw, J. P. (1994). Neutron diffraction reveals the site of amantadine blockade in the influenza A M2 ion channel. Virology 202, 287293.[CrossRef][Medline]
Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D. & Bienz, K. (2002). Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76, 59745984.
Ewart, G. D., Sutherland, T., Gage, P. W. & Cox, G. B. (1996). The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels. J Virol 70, 71087115.[Abstract]
Gibbs, J. S., Malide, D., Hornung, F., Bennink, J. R. & Yewdell, J. W. (2003). The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function. J Virol 77, 72147224.
Gonzalez, M. E. & Carrasco, L. (1998). The human immunodeficiency virus type 1 Vpu protein enhances membrane permeability. Biochemistry 37, 1371013719.[CrossRef][Medline]
Griffin, S. D., Beales, L. P., Clarke, D. S., Worsfold, O., Evans, S. D., Jaeger, J., Harris, M. P. & Rowlands, D. J. (2003). The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett 535, 3438.[CrossRef][Medline]
Halestrap, A. P. & Brennerb, C. (2003). The adenine nucleotide translocase: a central component of the mitochondrial permeability transition pore and key player in cell death. Curr Med Chem 10, 15071525.[Medline]
Harada, T., Tautz, N. & Thiel, H. J. (2000). E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J Virol 74, 94989506.
Hay, A. J., Wolstenholme, A. J., Skehel, J. J. & Smith, M. H. (1985). The molecular basis of the specific anti-influenza action of amantadine. EMBO J 4, 30213024.[Abstract]
Hsu, M., Zhang, J., Flint, M., Logvinoff, C., Cheng-Mayer, C., Rice, C. M. & McKeating, J. A. (2003). Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci U S A 100, 72717276.
Jacotot, E., Ferri, K. F., El Hamel, C. & 17 other authors (2001). Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2. J Exp Med 193, 509519.
Jubin, R., Murray, M. G., Howe, A. Y., Butkiewicz, N., Hong, Z. & Lau, J. Y. (2000). Amantadine and rimantadine have no direct inhibitory effects against hepatitis C viral protease, helicase, ATPase, polymerase, and internal ribosomal entry site-mediated translation. J Infect Dis 181, 331334.[CrossRef][Medline]
Kunkel, T. A., Bebenek, K. & McClary, J. (1991). Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol 204, 125139.[Medline]
Lamb, R. A. & Pinto, L. H. (1997). Do Vpu and Vpr of human immunodeficiency virus type 1 and NB of influenza B virus have ion channel activities in the viral life cycles? Virology 229, 111.[CrossRef][Medline]
Liljestrom, P., Lusa, S., Huylebroeck, D. & Garoff, H. (1991). In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol 65, 41074113.[Medline]
Lin, C., Lindenbach, B. D., Pragai, B. M., McCourt, D. W. & Rice, C. M. (1994). Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol 68, 50635073.[Abstract]
Loewy, A., Smyth, J., von Bonsdorff, C. H., Liljestrom, P. & Schlesinger, M. J. (1995). The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process. J Virol 69, 469475.[Abstract]
Martin, J., Navas, S., Fernandez, M., Rico, M., Pardo, M., Quiroga, J. A., Zahm, F. & Carreno, V. (1999). In vitro effect of amantadine and interferon alpha-2a on hepatitis C virus markers in cultured peripheral blood mononuclear cells from hepatitis C virus-infected patients. Antivir Res 42, 5970.[CrossRef][Medline]
Medeiros, R., Escriou, N., Naffakh, N., Manuguerra, J. C. & van der Werf, S. (2001). Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes. Virology 289, 7485.[CrossRef][Medline]
Meyers, G., Tautz, N., Becher, P., Thiel, H. J. & Kummerer, B. M. (1996). Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs. J Virol 70, 86068613.[Abstract]
Mizushima, H., Hijikata, M., Asabe, S., Hirota, M., Kimura, K. & Shimotohno, K. (1994). Two hepatitis C virus glycoprotein E2 products with different C termini. J Virol 68, 62156222.[Abstract]
Mottola, G., Cardinali, G., Ceccacci, A., Trozzi, C., Bartholomew, L., Torrisi, M. R., Pedrazzini, E., Bonatti, S. & Migliaccio, G. (2002). Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293, 3143.[CrossRef][Medline]
Newton, K., Meyer, J. C., Bellamy, A. R. & Taylor, J. A. (1997). Rotavirus nonstructural glycoprotein NSP4 alters plasma membrane permeability in mammalian cells. J Virol 71, 94589465.[Abstract]
Ohuchi, M., Cramer, A., Vey, M., Ohuchi, R., Garten, W. & Klenk, H. D. (1994). Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein. J Virol 68, 920926.[Abstract]
Okada, A., Miura, T. & Takeuchi, H. (2001). Protonation of histidine and histidine-tryptophan interaction in the activation of the M2 ion channel from influenza a virus. Biochemistry 40, 60536060.[CrossRef][Medline]
Pavlovic, D., Neville, D. C., Argaud, O., Blumberg, B., Dwek, R. A., Fischer, W. B. & Zitzmann, N. (2003). The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci U S A 100, 61046108.
Piai, G., Rocco, P., Tartaglione, M. T. & 7 other authors (2003). Triple (interferon, ribavirin, amantadine) versus double (interferon, ribavirin) re-therapy for interferon relapser genotype 1b HCV chronic active hepatitis patients. Hepatol Res 25, 355363.[CrossRef][Medline]
Piller, S. C., Ewart, G. D., Premkumar, A., Cox, G. B. & Gage, P. W. (1996). Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers. Proc Natl Acad Sci U S A 93, 111115.
Piller, S. C., Ewart, G. D., Jans, D. A., Gage, P. W. & Cox, G. B. (1999). The amino-terminal region of Vpr from human immunodeficiency virus type 1 forms ion channels and kills neurons. J Virol 73, 42304238.
Pinto, L. H., Holsinger, L. J. & Lamb, R. A. (1992). Influenza virus M2 protein has ion channel activity. Cell 69, 517528.[Medline]
Rahmani, Z., Huh, K. W., Lasher, R. & Siddiqui, A. (2000). Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential. J Virol 74, 28402846.
Robertson, B., Myers, G., Howard, C. & 14 other authors (1998). Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. International Committee on Virus Taxonomy. Arch Virol 143, 24932503.[CrossRef][Medline]
Sakaguchi, T., Leser, G. P. & Lamb, R. A. (1996). The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus. J Cell Biol 133, 733747.[Abstract]
Sakai, A., Claire, M. S., Faulk, K., Govindarajan, S., Emerson, S. U., Purcell, R. H. & Bukh, J. (2003). The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences. Proc Natl Acad Sci U S A 100, 1164611651.
Salom, D., Hill, B. R., Lear, J. D. & DeGrado, W. F. (2000). pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus. Biochemistry 39, 1416014170.[CrossRef][Medline]
Saunier, B., Triyatni, M., Ulianich, L., Maruvada, P., Yen, P. & Kohn, L. D. (2003). Role of the asialoglycoprotein receptor in binding and entry of hepatitis C virus structural proteins in cultured human hepatocytes. J Virol 77, 546559.[CrossRef][Medline]
Schmidt-Mende, J., Bieck, E., Hugle, T., Penin, F., Rice, C. M., Blum, H. E. & Moradpour, D. (2001). Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 276, 4405244063.
Takada, S., Shirakata, Y., Kaneniwa, N. & Koike, K. (1999). Association of hepatitis B virus X protein with mitochondria causes mitochondrial aggregation at the nuclear periphery, leading to cell death. Oncogene 18, 69656973.[CrossRef][Medline]
Takeuchi, K. & Lamb, R. A. (1994). Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport. J Virol 68, 911919.[Abstract]
Thuluvath, P. J., Pande, H. & Maygers, J. (2003). Combination therapy with interferon-alpha(2b), ribavirin, and amantadine in chronic hepatitis C nonresponders to interferon and ribavirin. Dig Dis Sci 48, 594597.[CrossRef][Medline]
Tian, P., Estes, M. K., Hu, Y., Ball, J. M., Zeng, C. Q. & Schilling, W. P. (1995). The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum. J Virol 69, 57635772.[Abstract]
van Kuppeveld, F. J., Hoenderop, J. G., Smeets, R. L., Willems, P. H., Dijkman, H. B., Galama, J. M. & Melchers, W. J. (1997). Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release. EMBO J 16, 35193532.
Wang, C., Lamb, R. A. & Pinto, L. H. (1995). Activation of the M2 ion channel of influenza virus: a role for the transmembrane domain histidine residue. Biophys J 69, 13631371.[Abstract]
Weegink, C. J., Sentjens, R. E., Beld, M. G., Dijkgraaf, M. G. & Reesink, H. W. (2003). Chronic hepatitis C patients with a post-treatment virological relapse re-treated with an induction dose of 18 MU interferon-alpha in combination with ribavirin and amantadine: a two-arm randomized pilot study. J Viral Hepat 10, 174182.[Medline]
Yanagi, M., St Claire, M., Shapiro, M., Emerson, S. U., Purcell, R. H. & Bukh, J. (1998). Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 244, 161172.[CrossRef][Medline]
Zilly, M., Lingenauber, C., Desch, S., Vath, T., Klinker, H. & Langmann, P. (2002). Triple antiviral re-therapy for chronic hepatitis C with interferon-alpha, ribavirin and amantadine in nonresponders to interferon-alpha and ribavirin. Eur J Med Res 7, 149154.[Medline]
Received 8 September 2003;
accepted 6 November 2003.