key: cord-0713977-oflazi3s
authors: Haqshenas, G.; Dong, X.; Ewart, G.; Bowden, S.; Gowans, E.J.
title: A 2a/1b full-length p7 inter-genotypic chimeric genome of hepatitis C virus is infectious in vitro
date: 2007-03-30
journal: Virology
DOI: 10.1016/j.virol.2006.10.014
sha: 79d081da39e4c7399ceb62b3171514261a8d6e69
doc_id: 713977
cord_uid: oflazi3s

The p7 protein of hepatitis C virus (HCV) functions as an ion channel in planar lipid bilayers, and its function is vital for the virus life cycle. In this study, we replaced either the entire or partial p7 of genotype 2a (strain JFH1), an HCV strain that replicates and produces virus progeny in vitro, with the corresponding regions of the p7 protein from genotype 1b (Australian isolate, HCV-A). Compared to wild type, the chimeric viruses reached their peak of infectivity with a delay but they produced a comparable titer to the wild type virus and the progeny viruses were able to infect naive permissive cells. Amantadine treatment of wild type and chimeric viruses reduced the virus titers by about 50% and 45%, respectively. Therefore, in this study, for the first time, we demonstrated that genotype 2a (JFH1 strain) genome encoding a full-length genotype 1b p7 gene produces infectious particles in vitro. These chimeric viruses are valuable instruments for comparative studies of the p7 proteins.

Hepatitis C virus (HCV) is a major etiological agent of chronic hepatitis, which can lead to hepatocellular carcinoma and is currently the leading single indicator for liver transplantation in the developed world. The threat posed to the health systems in developed and developing countries by HCV infection continues to grow at an alarming rate. In the most recent report of WHO on HCV prevalence, it has been estimated that 123 million individuals are infected with the virus worldwide.

HCV is classified in the family Flaviviridae, genus Hepacivirus and is a small enveloped virus with a positive-sense single stranded RNA genome. The genome encodes a polyprotein that is co-and post-translationally cleaved into at least 10 structural and nonstructural (NS) viral proteins (Fields et al., 1996) . One of these, the p7 protein, is a small, membrane-associated protein of 63 amino acids that is located at the junction of the structural and NS proteins (Lin et al., 1994) . Thus far, it has not been determined if the p7 protein is a structural or an NS protein. The protein is comprised of two tails located in the lumen of the endoplasmic reticulum (ER), two hydrophobic transmembrane α-helices, TM1 and TM2, and an intervening basic loop located in the cytoplasm (Carrere-Kremer et al., 2002) . Using plasmid expression vectors, p7 has been shown to be mainly localized in the endoplasmic reticulum (Carrere-Kremer et al., 2002; Griffin et al., 2005) and possibly in mitochondrial membranes while a small percentage of the over-expressed p7 protein was detected in the plasma membrane (Griffin et al., 2004) . However, we have recently shown that p7 is localized to the ER, not mitochondria, in cells transfected with a replication-competent recombinant genome of the JFH1 strain of HCV (Haqshenas et al., in press ).

It has recently been shown that the p7 protein forms ion channels in planar lipid bilayers (Griffin et al., 2003; Pavlovic et al., 2003; Premkumar et al., 2004) . p7 belongs to a family of proteins known as viroporins, which homo-oligomerize to form ion channels in cellular membranes (Gonzalez and Carrasco, 2003) . Similar to that which has been shown for other viral ion channel forming proteins like the influenza virus proteins, M2 (Duff and Ashley, 1992; Duff et al., 1994; Pinto et al., 1992) , BM2 (Mould et al., 2003) , and NB (Sunstrom et al., 1996) , HIV proteins Vpu and Vpr (Piller et al., 1996) , alphavirus 6K protein (Melton et al., 2002) , Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV) E protein , GBV-B p7 (Premkumar et al., 2006) and Dengue virus M protein (Premkumar et al., 2005) , the p7 protein seems to have an essential role in the HCV life cycle. In an in vivo study, p7 was shown to be essential for HCV replication as viral RNA with the p7 region deleted or mutated was non-infectious in chimpanzees (Sakai et al., 2003) . However, p7 is dispensable for viral RNA replication as replicons which lack the p7 gene replicate efficiently (Blight et al., 2000; Lohmann et al., 1999) . Therefore, it is concluded that p7 is not required for efficient HCV genome replication, but is essential for the production of infectious virions (Sakai et al., 2003) .

Recently, the H77 (Yi and Lemon, 2004) and JFH1 strains of HCV genotypes of 1a and 2a, respectively, were demonstrated to replicate and produce progeny virus in cell culture systems (Lindenbach et al., 2005; Wakita et al., 2005; Yi et al., 2006; Zhong et al., 2005) . Very recently, chimeric JFH1 viruses encoding the genotypes 1a, 1b and 3a structural proteins, p7 and partial NS2 were also constructed Pietschmann et al., 2006) . Although these cell culture systems will play a significant role to increase our understanding of the molecular biology of HCV and drug discovery, there remains a need for a cell culture system to discover ion channel inhibitors and investigate their specific effects on the p7 protein.

Currently, there are no specific targeted antiviral agents effective against HCV. The current treatment for HCV is a combination of pegylated interferon-alfa and ribavirin, which is only effective in about 50% of patients. This therapy produces considerable side effects and can lead to the emergence of resistant virus strains (Fargion et al., 2004) . The p7 protein, as an ion channel forming protein, is a potential target for ion channel inhibitors. p7 ion channel activity in planar lipid bilayers is inhibited by hexamethylene amiloride (Premkumar et al., 2004) , long-alkyl-chain iminosugar derivatives (Pavlovic et al., 2003) and amantadine, which also inhibited p7 activity in an hemadsorption assay (Griffin et al., 2003; Griffin et al., 2004) . In clinical trials, amantadine has been shown to significantly increase the number of HCV-infected patients who showed a sustained virological response (Berg et al., 2003; Deltenre et al., 2004; Mangia et al., 2004) while other data have not supported these findings (Thuluvath et al., 2004) . These contrasting findings may be a reflection of amantadine resistance of the ion channel function of p7 found in some HCV strains (Steinmann et al., 2005) .

Collectively, there is an urgent demand for authentic cell culture systems to examine ion channel inhibitors on the p7 protein of different genotypes. Therefore, in the current study, we investigated if the JFH1 strain could be used as a backbone to generate chimeric viruses encoding the p7 protein of genotype 1b of HCV. Such chimeric viruses would be valuable tools to investigate the specific effects of ion channel inhibitors on the function of the p7 protein.

Before the chimeric viruses were synthesized, we examined the ability of HCV-A.p7 to form functional ion channels. As shown in Fig. 1B , following the addition of the full-length p7 peptide to the CIS chamber of the planar lipid planar rig, ion currents were detected after brief periods of stirring. This indicates that the p7 has incorporated and formed ion channels in the lipid bilayer separating the CIS and TRANS chambers. Similarly, it was found that the N-terminal 38 residues comprised of the lumenal tail, TM1, and the cytosolic loop could form ion channels (data not shown). These data are in agreement with the computer modeling showing that only TM1 is involved in the channel formation (Patargias et al., 2006) . As can be seen in Fig. 1C , amantadine at a final concentration of 100 μM did not block the ion channel activity of the protein.

Before the generation of the chimeric viruses, we compared the deduced amino acid sequences of the p7 of genotype 2a (JFH1) and 1b (HCV-A). The sequences shared about 60% identity in a pairwise alignment (Fig. 2) . Two chimeric JFH1 cDNA constructs were generated, one encoding the partial and the other one encoding the entire p7 gene of genotype 1b (Fig. 3) , in response to a previous report that the amino acid sequences of the lumenal tails of p7 protein were crucial for E2-p7 and p7-NS2 cleavage (Sakai et al., 2003) .

In order to demonstrate that the chimeric genomes were replication-competent, Huh7 cells were transfected with wild type and recombinant JFH1 genomes and examined 2 to 3 days post-transfection by IF with anti-core antibody. The lysates of the infected cells were also subjected to immunoblot analysis. As shown in Fig. 4A , cells transfected with the recombinant genomes showed an intense signal by IF. The positive cells showed a core-specific punctuated cytoplasmic staining pattern as described previously (Hope et al., 2002) . In Western blot analysis, a band with an approximate size of 20 kDa, consistent with the molecular weight of the core protein, was observed in the cells transfected with the wild type and chimeric genomes (data not shown). As the control GNDJFH1 mutants failed to generate any signal in either IF or Western blot, these data indicate that the recombinant genomes were replicationcompetent. Since the results generated from GNDJFH1 mutant were identical to those from G-Fp7 and G-Pp7, in the next steps, we only used GNDJFH1 as a control.

After transfection, the Huh7 cells were passaged to determine if they were persistently infected. After each passage, the cells were examined for core protein by IF. At the first passage, the proportion of core antigen-positive cells in the culture transfected with the wild type RNA slightly increased (from 40-50% to about 60-70%) while the proportion of positive cells in the culture transfected with the chimeric genomes decreased slightly (from 40-50% to about 30-40%, data not shown). High numbers of non-adherent cells were noted continuously in the culture transfected with the wild type RNA, while the Pp7 and Fp7-transfected cells failed to show a CPE until passage 3. This is consistent with the IF data showing that the percentage of core antigen-positive cells in the wild type-transfected culture remained at 90-100% until passage 5 and then declined sharply to 10-20%. The cells became totally negative by passage 6. To investigate if a virus-resistant cell subset dominated during the course of infection, we re-exposed the cells to the virus and showed that the cells were successfully re-infected (data not shown). In the Pp7-and Fp7-transfected cells, the proportion of positive cells declined to about 30% in passage 2 but increased significantly in passage 3 (about 70-80% positive, data not shown). About 30-50% of the cells were still positive for core antigen at passage 7 when the experiment was concluded. To determine if the supernatants of the cells were infectious, naive Huh7 cells were infected with the supernatant fluid containing the highest viral titers. Three days post-infection, the cells were examined by anti-core-antigen IF test and shown to be positive (Fig. 4A ). The lysates of the infected cells were also subjected to Western blot analysis and showed a band with an approximate size of 20 kDa in the cells transfected with the wild type and chimeric genomes (Fig. 4B) .

We also examined the viral RNA titer in the cell culture supernatant of each passage. As shown in Fig. 5A , the titer of the wild type viral RNA in the supernatant increased at passage one and remained high during the next passages before it gradually decreased to undetectable levels at passage 7. In the case of the chimeric genomes, the virus titer declined slightly in passage 2 but increased in passage 3-4 and remained at this level until the end of the experiment (Fig. 5A ). The chimeric viruses did not reach the peak titer of wild type at any time point showing that the chimera replicated with slower kinetics than the wild type. The maximum titers of the wild type JFH1, P-p7 and F-p7 that were achieved in this study were 7.4 × 10 7 , 3.6 × 10 7 and 3.5 × 10 7 viral RNA copies/ml, respectively. The specific infectivity of the wild type and F-p7 was calculated as 2900 and 1600 RNA copies per FFU, respectively. We also determined the infectivity titers of the wild type and the chimeric viruses of the supernatants up to passage 6 posttransfection. Consistent with the results from the viral RNA titers, the wild type virus reached its peak in early passages while Pp7 and Fp7 chimeras reached their highest infectivity titers at passage 3 and 5, respectively (Fig. 5B ). Immunoblot analysis of the cells transfected with wild type and chimeric genomes, and the cells infected with the wild type and chimeric viruses did not reveal a major difference in the pattern of proteins that reacted with the monoclonal antibody AP33 (Fig. 6 ). This suggests that the cleavage of E2 from its precursor protein was not affected by the insertion and did not change during passaging the transfected cells.

The heterologous p7 gene in the chimeric viruses is stable RNA viruses commonly introduce compensatory mutations in the heterologous genes to increase viral fitness. To investigate if any mutation occurred in the HCV-A.p7 genes in the backbone of JFH1, we examined the nucleotide sequences of Fig. 3 . Schematic representation of the wild type and chimeric JFH1 polyproteins. The p7 protein is comprised of two luminal tails (LT) and two transmembrane domains (TM1 and TM2) and an intervening cytosolic loop. Hatched boxes indicate the JFH1 p7 sequences that were replaced by the corresponding regions in HCV-A.p7 to generate the partial (P-p7) and complete (F-p7) JFH1/HCV-A.p7 chimeras. the nascent recombinant viruses. The viral RNA was extracted from the cell culture supernatant of the passage 5 culture and was subsequently used in a one step RT-PCR with genomespecific primers, FE2 and RNS2 (Table 1) . Agarose gel electrophoresis of the PCR products from the chimeras showed one distinct band with an electrophoretic mobility similar to that of the product from the wild type virus (Fig. 7A) . Nucleotide sequencing of the products did not reveal any mutations in the sequence encoding p7 and flanking regions in the chimeric viruses (Figs. 7B, C) .

It has been reported that the strain J4 of genotype 1b is sensitive to amantadine (Griffin et al., 2003) while the JFH1 strain of genotype 2a is resistant to this drug (Steinmann et al., 2005) . We investigated whether the full-length p7 chimera (F-p7) with an identical TM1 to strain J4 is sensitive to amantadine. As shown in Fig. 8, 30 μM amantadine reduced the wild type and chimeric virus titer in the supernatant at day 3 post-infection by about 0.3 and 0.25 log, respectively. This effect was not seen at day 1 post-infection and was not due to amantadine-induced cytotoxicity, as determined by the MTT assay (data not shown).

This study describes the generation of two novel chimeric viruses in which the partial or the entire p7 protein gene of the JFH1 strain of HCV was substituted with the corresponding gene from a genotype 1b virus (HCV-A). These novel chimeric viruses are valuable tools for comparative studies of the function of p7 in an authentic cell culture system and for the examination of the effects of potential ion channel inhibitors in vitro.

p7 protein is a small protein of HCV that is highly membrane associated. The protein is comprised of two lumenal tails, two TM domains with an intervening cytosolic loop. The lumenal tails at the N-and C-termini of the protein were previously reported to contain genotype-specific sequences since the chimeric 1a/2a virus was infectious only when the lumenal tail sequences of the backbone genome of genotype 1a were left unaltered in the chimeric genome (Sakai et al., 2003) . Differences in the amino acids of the N-terminal lumenal tails of genotype 1a (H77C) and genotype 2a (J6) p7 proteins are restricted to positions 4 and 9. In H77C, there are two polar amino acids of asparagines at positions 4 and 9 while in J6 there are two charged amino acids of lysine and histidine at the corresponding positions (Sakai et al., 2003) . The J4 p7, like the HCV-A.p7, contains two asparagines at positions 4 and 9 while JFH-1 p7, like J6 p7, contains a lysine and a histidine residue at the corresponding positions, respectively. Moreover, the differences between JFH1 and HCV-A in the C-terminal lumenal tail are greater than the differences between J6 and H77C (Sakai et al., 2003) . Nevertheless, in the present study, despite these differences, the full-length JFH1/HCV-A.p7 chimera was infectious and produced infectious particles. This might reflect the difference between an in vivo and an in vitro system to examine the chimeric HCV genomes. To further examine the possibility of the existence of genotype-specific sequences in the lumenal tails, the chimeric 1a/2a genomes can now be examined in an in vitro assay since a cell culture system is now available to grow genotype 1a and its chimeric viruses (Yi et al., 2006) . In this study, although the chimeric genomes produced infectious viruses with comparable titers to wild type, compared to the wild type, the chimeric viruses reached their peak of infectivity with a delay. Sequencing analysis of the insert regions did not reveal any compensatory mutation in the p7 and flanking regions. Moreover, the immunoblot analysis did not demonstrate a major difference in the processing of the E2-p7-NS2 precursor proteins of the wild type and chimeras following either transfection or infection. However, it is likely that a compensatory mutation occurred in the viral genome, outside of the p7 and flanking regions, which made the chimeric viruses more replication-competent.

Until recently, HCV studies were hindered by the lack of a robust cell culture system to grow the virus in vitro. Recently, Wakita et al. (2005) during the preparation of this manuscript, chimeric JFH1 viruses encoding the genotype 1a, 1b and 3a structural proteins, p7 and partial NS2 were also constructed Pietschmann et al., 2006) . Despite the importance of these reports, the lack of a cell culture system to support the replication of all the HCV genotypes remains a major obstacle to study different HCV genotypes and develop antiviral drugs that are effective against all genotypes. Moreover, in the current cell culture systems, it is very difficult to justify the specific effects of any ion channel inhibitors on the function of p7 protein. Therefore, in the present study, based on the JFH1 strain, we generated two novel chimeric viruses that encode only the genotype 1b p7 gene to facilitate the comparative study of the ion channel activity of the genotype 1 and 2 p7 proteins since genotype 1 is responsible for a high proportion of HCV-infected patients in most geographic areas. In most Asian countries, genotype 1b is predominant while in the United States and Europe genotype 1a and 1b are predominant (Bukh et al., 1995; Lau et al., 1996) . The ion channel activity of genotype 1b p7 protein (Yanagi et al., 1998) has been shown to be sensitive to amantadine (Griffin et al., 2004) . The amino acid sequence of the J4 isolate only differs in two amino acid positions to the HCV-A p7: one in the cytosolic loop and one in TM2 (Fig. 9 ). Using computer modeling and electron microscopy, it has been proposed that only TM1, but not TM2, is involved in ion channel formation (Carrere-Kremer et al., 2002; Griffin et al., 2003; Patargias et al., 2006) . We have also examined the Nterminal subunit of p7 for ion channel activity and confirmed the previous reports by showing that the N-terminal subunit of p7 per se, independent from the C-terminal subunit, forms ion channel in an artificial lipid bilayer (unpublished data). Furthermore, J4 p7 has been shown to be sensitive to amantadine in an artificial lipid bilayer and also in an in vitro system (Griffin et al., 2003; Griffin et al., 2004) . Computer modeling of the p7 protein of J4 strain demonstrated that amantadine resides within the pore, formed by oligomerization of TM1, in the vicinity of His-17 and interacts with Ser-21 via hydrogen bonding (Patargias et al., 2006) . Therefore, we examined the sensitivity of the full-length chimeric virus, containing an identical Nterminal subunit to the J4 strain p7 protein, for sensitivity to amantadine. This study demonstrated that amantadine has a similar effect on the life cycles of the chimeric (encoding the full- Fig. 7 . The p7 proteins in the chimeric viruses are stable. (A) The RNA was extracted from passage 4 of the wild type and the chimeric viruses containing partial p7 (Pp7) and the entire p7 (Fp7) proteins and subjected to RT-PCR amplification. The PCR products were visualized in a 1% agarose gel. (B and C) The PCR products were then sequenced, and the depicted amino acid sequences have been shown. length HCV-A.p7) and wild type viruses as determined by the titer of the virus secreted into the supernatant. However, a much larger reduction in the titer of the chimeric virus was expected, due to the previous published data (see above). We also demonstrated that the ion channel activity of the HCV-A (genotype 1b) p7 protein was resistant to amantadine in artificial lipid bilayers. The discrepancy between our results on HCV-A (genotype 1b) and the published data on J4 p7 (genotype 1b) may suggest a role for the cytosolic loop and TM2 in the response to amantadine. To examine this, we are currently in the process of changing the two amino acids to convert HCV-A.p7 to J4 p7. Although the results of the amantadine effect on JFH1 wild type are in contrast to previously published data (Steinmann et al., 2005) , they are in agreement with the more recently published data showing that amantadine reduced the number of JFH1 foci by about 70% in cell culture (Boume et al., 2005) . The mechanism of the effect of amantadine on the HCV life cycle is not clear yet. However, the results presented in this manuscript suggest that this mechanism is far more complex than simply blocking ion channel pores. It has been documented that the infection of cells with porcine circovirus type 2 that does not contain any ion channel forming protein is inhibited by amantadine through inhibition of clathrin-mediated endocytosis (Nauwynck et al., 1999) . HCV, like porcine circovirus type 2, enters the cells via clathrin-mediated endocytosis (Blanchard et al., 2006) and, therefore, the effect of amantadine on the HCV life cycle might partially be through the blocking of this function. Collectively, because the only difference between the wild type and the chimera is the p7 protein and irrespective of the sensitivity of p7 to amantadine, the drug has almost the same effect on the viral life cycle, it is possible that this effect is not through the inhibition of p7 protein.

In conclusion, we believe that the novel chimeric viruses described in this manuscript are valuable tools for the study of the function of the p7 protein and also to specifically examine the effects of potential ion channel inhibitors in a genuine cell culture system.

Huh7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco/BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (P.A. Biologicals Co., Melbourne, Australia), 100 IU of penicillin/ml and 100 μg of streptomycin/ml. The wild type and replication-defective mutant full-length JFH1 cDNA constructs were kindly provided by Dr. Wakita . The full-length Australian HCV cDNA (HCV-A) was constructed in our laboratory (Trowbridge and Gowans, 1998) . E. coli strain JM109 (Promega, Madison, Wis) was used in all transformation experiments.

A synthetic p7 peptide, based on the sequence of HCV-A, was synthesized at GenScript Corporation (NJ, USA) and tested in an planar lipid bilayers, as described previously (Premkumar et al., 2004) , to determine if it has ion channel activity. Similarly, a peptide was synthesized corresponding to the N-terminal 38 Fig. 9 . Comparison analysis of the deduced amino acid sequences of p7 proteins of J4 and HCV-A isolates of HCV. TM1 and TM2 are underlined (Sakai et al., 2003) . residues (ALENLVVLNAASVAGAHGILSFLVFFCAAW-YIKGRLVP) and was also examined for ion channel activity. Amantadine sensitivity of the p7 ion channel was determined by using 100 μM amantadine as described before (Premkumar et al., 2004) .

The full-length wild type and defective GND mutant of JFH1 were digested with XbaI and NotI restriction enzymes, blunt ended and re-circularized using standard protocols (Sambrook et al., 1989) . The recombinant plasmids were called wtJFH-N and GJFH-N, respectively. Two chimeric JFH1/HCV-A.p7 fragments were then synthesized by fusion PCR, using the primers shown in Table 1 . In the first chimera, the entire p7 gene of JFH1 was substituted by the HCV-A.p7. In the second, the partial p7 protein (TM1, TM2 and cytosolic loop) of JFH1 was replaced by the corresponding region of HCV-A to ensure that the lumenal tails of the p7 protein of JFH1 remained unchanged. The chimeric fragments were then digested with SphI and cloned into the wtJFH-N and GJFH-N plasmids digested with the corresponding enzyme to create 5′JFH-Fp7 and 5′JFH-Pp7 (containing the full-length and partial HCV-A.p7 genes), respectively. These constructs were then digested with EcoRI and KpnI, and the released fragments containing IRES, core, E1, E2, partial or full-length HCV-A.p7 and NS2 were cloned into wtJFH1 and mutant GNDJFH1 (digested with the corresponding enzymes). The resultant recombinant JFH1 constructs were named J-Fp7 (JFH1 encoding full-length HCV-A.p7), G-Fp7 (GNDJFH1 encoding full-length HCV-A. p7), J-Pp7 (JFH1 encoding partial HCV-A.p7) and G-Pp7 (GNDJFH1 encoding partial HCV-A.p7). The nucleotide sequences of the fragments were confirmed by using FE2 and RNS2 primers (Table 1) in an automated cycle sequencing reaction.

In vitro RNA transcription and transfection RNA transcripts were synthesized as described (Huang et al., 2005) . To examine the replication competency of the recombinant genomes, 4 μg of in vitro generated RNA transcripts was transfected into cells in 6-well plates (Nalge Nunc, Rochester, NY) using 10 μl of DMRIE-C (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The transfection mixture was removed 5 h later, the cells washed twice with DMEM and complete medium added. The cells were trypsinized and passaged every 3-4 days for 7 passages as described previously (Zhong et al., 2005) .

The transfected and infected cells were fixed with cold acetone for IF analysis then stained with goat anti-HCV core antibody (ViroStat, Portland, ME) followed by Alexa Fluor 488-conjugated donkey anti-goat (Molecular Probes). The cells were then counterstained with propidium iodide and examined with a BioRad confocal microscope.

For immunoblot analysis, the cells were directly lysed in 2× Laemmli buffer (BioRad, Hercules, CA). Goat anti-HCV core antibody and the monoclonal antibody AP33 (Owsianka et al., 2005) were used to detect the HCV core and E2 proteins, respectively. ECL Western blotting detection reagents (Amersham Pharmacia Biosciences) were used according to the manufacturer's instructions to detect the immunocomplexes.

The supernatant of each passage was filtered through a 0.45 μM filter and stored at − 70°C. From each passage, 75 μl of the supernatant fluids was collected and 50 μl of aliquots used for quantification. HCV viral loads were determined using the Versant HCV RNA 3.0 bDNA assay (Bayer HealthCare, Tarrytown, NY) according to the manufacturer's instructions.

Overnight cultures of Huh7 cells were treated with amantadine (Sigma) at final concentrations of 30 μM for 1 h that was not toxic for the cells in an MTT-based cytotoxicity assay (data not shown). The cells were then infected with the wild type and chimeric JFH1 samples containing 3.5 × 10 6 and 2.5 × 10 6 RNA copies/ml, respectively, in the presence of amantadine for 5 h. The cells were washed twice with DMEM, and fresh medium containing 30 μM amantadine was added. To determine whether amantadine has any effect on the progeny virus production, the cells were grown for 3 days and 200 μl samples collected on day 1 and 3 post-infection for quantification of HCV RNA. After each sampling, a same volume of fresh medium containing amantadine was added to each culture.

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This study was supported by grants from the National Health and Medical Research Council of Australia (NHMRC) and the Australian Centre for HIV and hepatitis virology (ACH2). We thank the late Dr. Alan R. Hibbs for his help in immunofluorescence studies and Dr. Wakita for the gift of cDNA plasmids of wild type and GND mutants of JFH1. We would like to thank Dr. Arvind Patel (University of Glasgow, UK) for providing us with AP33 monoclonal antibody and David Tyssen for his help in the data analysis. The HCV laboratory in the Burnet Institute is a member of ACH2 and the Australian Centre for Hepatitis Virology, Inc.