Correspondence Saba Khaliq, Functional and Applied Genomics Laboratory, National Center of Excellence in Molecular Biology, University of Punjab, Lahore 53700, Pakistan Tel: +92 300 4531036 e-mail: email@example.com
p7, a 63-residue peptide encoded by hepatitis C virus (HCV), a major pathogen associated with a risk of developing severe liver disease, is involved in ion channel activity in lipid bilayer membranes both in in vitro and cell-based assays. p7 protein consists of two transmembrane α-helices, TM1 and TM2 connected by a loop oriented towards the cytoplasm. HCV relies on p7 function in addition to ion channel formation for efficient assembly, release and production of infectious progeny virions from liver cells. p7 activity is strictly sequence specific as mutation analysis showed the loss of ion channel function. Moreover, p7 ion channel activity can be specifically inhibited by different drugs suggesting the protein as a new target for future antiviral chemotherapy. In the present review, we focused to bring together the recent development to explore the potential role of p7 protein in HCV infection and its inhibition as a therapy.
Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, which can lead to cirrhosis, liver failure and hepatocellular carcinoma throughout the world (1, 2). Like other RNA viruses, HCV possess a high degree of sequence variability that likely contributes to its ability to establish chronic infections after a mild acute phase. The best available current chemotherapeutic treatment comprises a combination of high-dose pegylated interferon alpha (IFN-α) with the guanosine analogue ribavirin (Rib) associated with severe side effects and can lead to the emergence of resistant virus strains (3–5). The efficacy of this regimen is largely dependent on the viral genotype; the most prevalent genotype 1 viruses possess high levels of innate resistance to IFN while resistance in other genotypes because of the highly variable nature of HCV (6, 7). Vaccine development is hindered by the lack of good in vitro and in vivo models of infection, the antigenic heterogeneity of the virus and its ability to avoid immune defenses.
The HCV is an enveloped positive-stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family with six major genotypes and approximately 100 subtypes depending on the geographical distribution of the virus (8). HCV genome encodes a single polyprotein precursor of approximately 3000 amino acid residues replicated in the cytosol through a negative-strand intermediate. An internal ribosome entry site (IRES) drives translation of the polyprotein, which is co- and post-translationally processed by cellular and viral proteases to yield mature viral structural proteins Core, E1 and E2, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, while an additional protein can be produced by a ribosomal frameshift in the N-terminal region of the polyprotein (9–11) (Fig. 1A). Core along with two envelope glycoproteins E1 and E2, released from the polyprotein by the endoplasmic reticulum (ER) signal peptidases of the host cell are the structural constitutes of the virus particle together with the host-derived lipid bilayer. The nonstructural proteins, released from the polyprotein after cleavage by HCV proteases NS2-3 and NS3/4A form a ribonucleoprotein complex with the virus genome that associates with intracellular membranes and is the site of RNA replication mediated by the NS5B protein (12, 13). Moreover, viral insensitivity to IFN/Rib maps to regions within the NS proteins that confer resistance to the innate immune response (14, 15).
A small hydrophobic protein (p7) located between the structural and nonstructural region of HCV is not clearly identified as structural or nonstructural protein and is shown to be an integral membrane protein (16–19). The identified function for p7 is an oligomeric ion channel formation capable of mediating cation flow across artificial membranes (20, 21). The recent in vivo experiments reported that p7 is essential for infection, while subgenomic HCV replicons do not contain p7, demonstrating that it is not necessary for RNA replication (22, 23). Although there are studies on the structure and function with biological and biochemical data on HCV p7 protein, the precise role/importance of p7 in the HCV life-cycle is difficult to determine. Moreover, given the importance of other ion channel-forming proteins for virus replication, the specific inhibition of HCV p7 presents an opportunity for a new antiviral therapy. The current review was attempted to compile in the light for the need of available information about HCV p7 gene at a single place.
Processing/generation of p7 gene
Grakoui et al. (24) in 1993 reported at least two distinct forms of N-deglycosylated E2-specific proteins and suggested that an additional cleavage product might be present between E2 and NS2 proteins (24). Lin et al. (17) identified this protein called as p7, by expression of a series of C-terminally truncated polyprotein and by fusion to a human c-myc epitope tag, which allowed isolation of the cleavage product and N-terminal sequence determination. The cleavage between p7 and NS2 is mediated by the ER signal peptidases of the host cell (17, 18, 25). Most cleavages in the polyprotein precursor occur during or immediately after translation but cleavages at the E2/p7 and p7/NS2 sites are delayed resulting in the production of an E2-p7-NS2 precursor protein (26). The presence of hydrophobic potential signal/anchor sequences preceding the E2/p7 and p7/NS2 cleavage sites and the results of cell-free translation analyses indicate that host signal peptidase may catalyse both of these cleavages with the presence of a discrete E2-p7-NS2 precursor and a reasonably stable E2-p7 species, suggesting that these cleavages are not cotranslational. Furthermore, incomplete cleavage at the E2/p7 site results in the production of two stable E2-specific proteins with different C termini, E2 and E2-p7 (17, 18, 26). Similarly the most closely related GB virus B (GBV-B) of flaviviridae contains a p13 ion channel protein (63-amino acid long peptide) between the two envelope proteins and NS2, with 25–33% amino acid homology (27). It is not known whether E2-p7 performs any specific function or not, however, the E2-p7 protein of bovine viral diarrhoea virus (BVDV) was not essential for cell culture infectivity because RNA transcripts from a bicistronic construct in which E2 and p7 were expressed separately could produce infectious virus (28). It has been suggested that HCV E2-p7 precursor may regulate the generation of native p7 and thus the formation of ion channel complexes, and may provide a means of including p7 in virions (29). Furthermore, in contrast to the other HCV structural proteins, the signal peptidase responsible for the E2-p7 cleavage appears to be unique to mammalian cells and is absent from insect cell systems, suggesting that the virus may have evolved to regulate this cleavage event through specific pathway to achieve specific E2/E2-p7 ratios (29) (Fig. 1B).
p7 an integral protein/localization of p7
The HCV virus particles assembly has been suggested to occur in the ER membranes like other enveloped viruses where it occurs in the host cell membrane (30), and different proteins including p7 play a central role in viral particle formation and budding. Like other viroporins such as GBV-B, p13 protein C and N terminals are oriented towards the ER lumen with the short segments that connect to the transmembrane domains (TMDs) (27). The C-terminal helix of p7 may also direct the localization of the protein itself or be part of such signals. After amino acid sequence analysis and structure predictions, the topology of p7 and its subcellular localization were predicted showing a fraction of p7 as an integral membrane polypeptide. p7 is a membrane-spanning protein found in the ER lumen that crosses the plasma membrane twice and has its N and C termini oriented towards the extracellular environment with signal sequence at the C-terminal TMD (16). p7 has two hydrophobic TMDs spanning amino acids 19–32 and 36–58 connected by a short cytoplasmic loop. It also has been demonstrated that the C-terminal TMD of p7 can function as a signal sequence that most likely promotes the translocation of NS2 into the ER lumen for appropriate cleavage by host signal peptidases. Moreover, p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway (16) (Fig. 2). Contrary to this investigation, colocalization of p7 with fluorescent markers showed a punctate staining pattern for cellular organelles indicating that it largely accumulated in membranes associated with the mitochondria of transfected cells. The localization of p7 was unaffected by the mutation in transfected 293 T cells (31). But in the proceeding work, the localization of p7 in the mitochondrial membrane was questioned and suggested a mitochondrial-associated ER localization as when a N-terminal-tagged p7 was over expressed in vitro from an expression vector, the protein was detected in mitochondria, but not in the ER, whereas if the protein was tagged at the C terminus it was detected in the ER (32). Moreover, Griffin et al. (32) reported that trafficking of HCV p7 is a complex process potentially regulated by both the cleavage from its upstream signal peptides and targeting signals within the protein sequence (32). HCV nonstructural proteins have also been observed to partially localize to ER cisternae around the mitochondria in replicon cells (33). It has been recently demonstrated that the p7 protein in the context of the full-length polyprotein encoded by a replication competent genome is only localized to the ER and has a possible role in HCV particle formation (34).
The recognition of virus proteins capable of enhancing membrane permeability has led to the description of a new family of proteins called viroporins to form ion channels in lipid bilayers of cells (35). Structurally, viroporins are generally 60–120 amino acid small proteins that homo-oligomerize to form hydrophilic cation channels after insertion into a lipid membrane to create ion channel with at least one membrane-spanning domain and tend to form oligomers involved in virus assembly or entry/exit, although many have additional functions (36–45). Based on the predicted structural, topological features and ion channel activity of HCV p7, it is proposed to include this protein in the viroporin family, as suggested for pestivirus p7 (20, 28). Premkumar et al. (46) in 2004 have confirmed that HCV p7 forms ion channels in planar lipid bilayer membranes. As reported previously, the ion channels had a very variable conductance and this is generally found when channel-forming virus proteins are inserted into planar bilayer membranes (47–50). Ion channels formed by p7 had a variable conductance; some channels had conductances as low as 14 pS. The reversal potential of currents flowing through the channels formed by p7 showed that they were permeable to potassium and sodium ions and less permeable to calcium ions. Addition of Ca+ to solutions made channels less potassium- or sodium-selective indicating the cation selectivity of channels with an assumption that the calcium ions interact with p7 and modify the structure of the channels, affecting the selectivity filter (46). This Ca+ selectivity is a characteristic reported for other viroporin too like M2 of influenza virus and human immunodeficiency virus (HIV)-1 Vpu protein (51). The cation selectivity of VPU is attributed to a ring of serine residues pointing towards the interior of the pore, while in HCV p7 serine amino acid at position 24 belongs to the putative residue lining the ion-conducting pore (52, 53). It has been reported that p7 can substitute for M2 of influenza virus in a cell-based functional assay measuring the transport of the influenza A haemagglutinin (31).
Structure of p7 ion channel
Although biological and biochemical data have been accumulated on most HCV proteins, the structure and function of the 63-amino acid p7 polypeptide of this virus have been not yet cleared. p7 forms ionic channels in the membrane, probably forming a hexameric or heptameric structure. Similar to other viral ion channels such as influenza virus M2 and HIV-1, Vpu secondary structure predictions suggest that folding of p7 could begin by forming two TM helices across the bilayer followed by stabilization of the monomeric protein and finally the oligomerization of the monomer to form the hexameric assembly through interaction of the helices connected by short loop (16, 20, 54). The loop is assumed to face the cytoplasm with the N and C termini facing the ER lumen. Recent electrophysiological experiments suggest that the N-terminal helix lines the pore (55). The conserved positively charged loop, its location on the surface of the membrane, its tendency to oligomerize in the presence of phospholipids as well as its specific interaction with phospholipid head groups could be the driving force of the protein oligomerization essential for the formation of the active ion channel. In addition, residues present in the TM α helices are responsible for the stable formation of oligomers (16). Griffin et al. (20) in 2003 demonstrated that recombinant HCV p7 proteins could be cross-linked as hexamers in HepG2 cells and that Escherichia coli-expressed p7 also formed hexamers in vitro, suggesting that this protein could be responsible for ion transport from the ER into the cytoplasm of HCV-infected host cells (20). p7 oligomerization has also been investigated in studies by Griffin et al. (31) and Clarke et al. (56), in both studies, di-thio-bis-succinimidyl proprionate was used as a crosslinking agent with hexahis-p7 and FLAG-p7 to form hexa- and heptamers, respectively, in a unilamellar lipid environment. It has also been suggested that p7 channels are heptameric assemblages with a predicted structure whereby the lumen is formed by the amphipathic amino-terminal TM helices (54, 56). Carboxy-terminal helices are thought to interact with adjacent p7 protomers, serving to stabilize the channel structure. In addition, the basic loop may form a constriction at one end of the channel, possibly serving to mediate channel gating (20). Accordingly, residues within the loop or the amino-terminal helix would be most likely to mediate channel opening and/or drug binding. On the basis of electron microscopy and biochemical data, the channel is proposed to be made by a hexameric bundle with a putative pore diameter of 3–5 nm (20). By mapping the amino acids of p7 onto the backbone of two α-helical domains of bacteriorhodopsin, a structure of a channel with a 2.3 nm pore diameter was predicted. In this model, the amphipathic channel forming the N-terminal helix (TM1) is stabilized by the C-terminal helix (TM2) and the adjacent N-terminal helix of the next monomer (20). Recently, a pure computational hierarchical methodology was applied for the modelling of the homo-oligomeric assembly of p7 channel-forming protein in the absence of any high-resolution structural and other biochemical data regarding the TM stretches. The results suggested a hexameric assembly with histidines involved in ion channel gating in respect of its structure and energy (54).
The HCV p7 ion channel is one of the smallest sized objects to be visualized by single particle image analysis to date. Luik et al. (57) in 2009 reported the 3-dimensional (3D) structure of HCV p7 ion channel (57). For the p7 channel, the detergent-solubilized hexameric protein content accounts for 42 kDa; the estimated mass of the negatively stained detergent-surrounded complex was ≈90 kDa. The 3D structure of a full-length viroporin was determined in which orientation of the p7 monomers within the density was established using immunolabelling with N and C termini-specific Fab fragments at a resolution of ≈16 Å revealing a flower-shaped protein architecture with protruding petals oriented towards the ER lumen, presenting a comparatively large surface area and providing potential interaction sites for cellular and virally encoded ER resident proteins. Moreover, inspection of 3D structure suggests that direct contacts between p7 monomers will be restricted to the ‘bottom’ half of the channel. There is a strong tilt angle of the density map observed at approximately half the channel's height (57). This structure shows that a much larger exposed surface area provided by the open petals may be available for interactions with host or viral proteins that were assumed based on computer models alone (31, 54).
Solid-state nuclear magnetic resonance (NMR) is well suited for studying membrane proteins in phospholipid bilayers (58). Magnetically aligned bicelle samples provide a unique opportunity to determine the 3D structure of proteins such as viroporins in their native environments. The orientation dependence of the resonance frequencies is especially informative about the structural details of the residues in TM helices, which are responsible for their ion channel activity and for binding to channel-blocking drugs. Initial solid-state NMR studies of p7, obtained from one p7 full-length protein and a truncated protein expressed in E. coli, in 14-O-PC/6-O-PC bicelles indicate that the protein contains helical segments that are tilted approximately 10 and 25° relative to the bilayer normal. A truncated construct corresponding to the second TMD of p7 is shown to have properties similar to those of the full-length protein and was used to determine that the helix segment tilted at 10° is in the C-terminal portion of the protein (59). Proceeding this work Cook et al. (60) performed NMR studies with milligram quantities of isotopic labelled p7 protein obtaining from full-length p7 and two truncated constructs in E. coli using a fusion partner, which directs the over-expression of protein in the inclusion bodies. The purified p7 polypeptides by exclusion chromatography were resolved in solution NMR two-dimensional HSQC spectra of uniformly N-labelled p7-polypeptides in DHPC isotropic micelles and same results were observed with same polypeptides in magnetically aligned 14-O-PC/6-O-PC bicelles into planar phospholipid bilayers (60). Recently, p7 strain that was chemically synthesized and purified was used for structure analysis by circular dichroism analysis, which revealed the p7 structure mainly as α-helical, irrespective of membrane mimetic medium, whereas the secondary structure elements of the monomeric form of p7 was revealed by NMR in triflouroethanol–water mixtures. The combined data of both approaches predicted the presence of a turn connecting as unexpected N-terminal α-helical to TM1 and along cytosolic loop bearing the dibasic motif and connecting TM1 and TM2 (53). Compared with the NMR data, HIV-1 Vpu ion channel p7 has quite different architecture, as Vpu has one TM helix and p7 has two, in addition there are significant differences in the structure and dynamics of internal loop and terminal regions of the two proteins (61).
The present available predicted channel structure of p7 showed that it forms a hexameric structure while more information will definitely aid the understanding of its function as an ion channel, an interaction partner for virally and host cell encoded proteins and as an antiviral drug target (Fig. 3).
p7 role in virus infectivity
The p7 protein appears to have an essential role in the HCV life cycle for virus assembly and release, as demonstrated for other members of viroporin family, like the influenza virus M2 protein (43, 62). Although not absolutely essential, viroporins greatly facilitate virus assembly and release in the viral replication cycle. HCV p7 is apparently not important for RNA replication as subgenomic replicons without p7 replicates efficiently in Huh-7 cells (22, 24, 63). The p7 proteins of HCV and BVDV most likely are required for virus assembly and/or release of infectious virus particles like other viral proteins with similar properties (28, 64). Similarly, HIV-1 viroporin, Vpu promotes HIV-1 infectivity and release of infectious virus particles through protein–protein interactions (65). In addition to the p7 polypeptide of BVDV, HCV closest relative hepacivirus GBV-B is also supposed to be crucial for virus replication (66). Moreover it has been reported that the charged residues of HCV p7, BVDV and GBV-B p13 are mainly responsible for the infectivity of the virus because these charges contributes to maintain the membrane topology of these proteins (23, 28, 66). It has also been observed that the p7 protein itself and not the primary sequence is critical for infectivity because the primary sequence encodes amino acids 779 and 781, the only two positions mutated exhibiting significant heterogeneity at all six nucleotide positions among different isolates of HCV (67). Supporting this, the p7 sequence of HCV apparently does not contain important RNA secondary structures (68). In chimpanzees, p7 was found to be essential for HCV infectivity as disruption of either uncleaved E2-p7 or p7-NS2 using an encephalomyocarditis virus IRES abolished infectivity in this system (23).
Apart from its ion channel activity, it has been suggested that p7 may play its role in virus replication through interaction with other viral or cellular proteins (23). In a naturally occurring intertypic chimera of HCV, the 5′ end of the genome including the sequence encoding the first 139 amino acids of NS2 was from genotype 2k, whereas the remainder of the genome was genotype 1b suggesting that the interaction of p7 could be with the 5′ UTR, C, E1, E2 and/or NS2 sequences (69). Evidence from the HCV culture system implies that both p7 and NS2 undergo critical protein–protein interactions during virus assembly (70). Yi et al. (71) demonstrated that interactions between NS2, E1 and p7 are essential for virus assembly and/or release and that each of these viral proteins plays an important role in this process. This protein–protein interaction was postulated to be important for anchoring p7 to NS2 within the membrane potentially enhancing the ability of the p7 to protect newly assembled particles; this hypothesis is consistent with the ability of the compensatory mutations to enhance the specific infectivity of particles released by the cell (71). Moreover, it has been reported that the p7 protein is crucial for late steps in virus assembly and release as upon transfection of viruses with several different p7 mutations (inactivating cis-active RNA packaging signals) a much higher ratio of cell associated to free virus particles as were observed suggesting it as an important virulence factor that may modulate fitness and in turn virus persistence and pathogenesis (72). On the other hand, based on these results it is concluded that interactions of p7 with other viral structural proteins and/ or NS2 are important for p7 to function in promoting virus assembly and release. The stimulatory effect of p7 on virus production is governed by genotype-specific sequences of other viral proteins implying that genotype-specific interactions of p7 govern the efficiency of assembly through interaction with the core protein too (72, 73). For the analysis of p7 importance in viral assembly, recently Brohm et al. (74) developed a transcomplementation assay to avoid the secondary effects caused by aberrant polyprotein cleavage, in which p7 defective HCV genomes were studied by providing E2, p7 and NS2, or in some cases p7 alone in a transient complementation assay as well as in stable cell lines, which all compensated the HCV morphogenesis, indicating the essential role of p7 in infectious virus production. This assay allowed the study of p7 function as an absolutely required protein for HCV production independent of additional/indirect effect of HCV polyprotein in comparison with other viroporins like influenza virus M2 and HIV-1 Vpu both of which facilitate infectious virus release but are not absolutely required for virus production (75). Moreover it was observed that p7 functions independently of an upstream sequence and tyrosine residue close to the conserved dibasic motif of p7 in virus production (74).
Importance of sequence in p7 function
The p7 exhibits significant genetic heterogeneity among isolates belonging to different genotypes. Sakai et al. (23) in 2003 examined that p7 of HCV contains genotype-specific sequences interacting with sequences of other regions of the genome. The C-terminal TMD of p7 is a signal sequence and the amino- and/or carboxyl-terminal tails of p7 contain sequences with genotype-specific function (23). The C-terminal helix of p7 has been shown to be capable of acting as a signal peptide when fused to the HCV E1 protein, facilitating correct glycosylation of the protein in HepG2 cells (16). In addition, genotype 1a and 1b p7 signal peptides appear to cleave with different efficiencies as determined by their amino acid sequences (76). It is possible that this process plays a role or may be a consequence of targeting p7 to different membrane compartments.
p7 sequences contain clusters of hydrophilic amino acids predicted to line the channel lumen suggesting that these might determine channel opening and drug sensitivity. As at least one function of p7 may be analogous to that of influenza virus M2 during particle secretion (31), it is possible that sequence variation might determine the sensitivity of HCV to p7 inhibitors. Region 771–788 coincides with the p7 loop linking the two TM helices of the protein having highly conserved loop and seems to be necessary for homodimerization and channel forming (31). The biological roles of p7 can be modulated by membranes; Perez-Berna et al. (77) in 2008 carried out the analysis of the membrane-active regions of p7 by observing the effect of a p7-derived peptide library from HCV (strain HCV_1B4J) on the integrity of different membrane model systems and identified a membranotropic region in p7 located at the loop domain (p7L) of the protein, characterizing its binding and interaction with model membrane systems and binding-induced structural changes of the peptide and the phospholipids. Furthermore, peptide p7L exhibits a high tendency to oligomerize in the presence of phospholipids, which could be the driving force for the formation of the active ion channel (77).
Meshkat et al. (78) in 2009 compared the p7 sequences from different HCV genotypes and identified an amino acid sequence homologous to the influenza virus M2 HXXXW/Y motif in several HCV genotypes using reverse genetic analysis. This sequence locates to the N-terminal subunit of p7 encompassing TM1 and cytosolic loop, which has been demonstrated as having ion channel activity previously (72). A conserved HXXXW/Y sequence was identified in genotypes 3, 4 and 5 when different p7 sequences were compared while the histidine residue of this sequence that is conserved in genotypes 1, 3, 4 and 5 is substituted by asparagine and glutamine in genotype 2 and 6 respectively (78). The structure of the M2 channel differs from that of p7; an M2 tetramer forms a functional channel, whereas a heptamer of p7 was reported to constitute the ion channel in artificial lipid membranes (56).
Effect of mutations on hepatitis C virus p7 activity
Little is known about how primary sequence governs p7 channel activity or drug interactions although several mutations have been shown to affect particle secretion in culture (79, 80), including the conserved basic charges on the cytosolic loop, which are known to be required for p7 activity in surrogate cell systems (31). The sequence, IKGR in genotype 1b, is rarely changed in other HCV isolates and, the K and R at positions 33 and 35, respectively, are absolutely conserved except in a few cases where the two are exchanged suggesting that the presence of charged residues at this locus is important for HCV p7 function. Mutation of the conserved cytosolic loop located between the TM α helices of HCV p7 (KR mutant protein) abrogated ion channel function without any effect on the intracellular localization of the protein (31). A HCV p7 hexamer model predicted that the K and R side-chains of each monomer project into the lumen, constricting the aperture on the cytosolic side of the channel, perhaps forming a gate controlling the flow of ions (20). Mutation of the dibasic K33–R35 motif to alanine effected the channel activity in liposomes while similar mutations block HCV particle production in culture and replication in chimpanzees as well as p7 ion channel activity in cellular assays (23, 31, 79, 81). The mutation of P49A caused a specific ion channel defect under standard conditions affecting channel stability and titration of increasing amounts of protein revealed a sudden switch of P49A protein to a hyperactive channel compared with the wild-type protein suggesting that Pro49 may regulate the formation of channel complexes. Pro49 is predicted to form a kink in the carboxy-terminal TMD, the orientation of which could conceivably regulate the formation of a stable structure (81).
Mutation in the conserved loop region in BVDV has been shown previously to affect virus infectivity in culture (28). Steinmann et al. (72) in 2007 have shown that interestingly replacement of the two conserved basic amino acids, Lys779 and Arg781, pertaining to the p7 loop dramatically suppressed the production of infectious viruses, highlighting the importance of this conserved motif as well as the relevance of the loop. Significantly, two other amino acids pertaining to the p7L peptide, Trp776 and Tyr788, are also essential for the p7 function (72). Interestingly, Sakai et al. (23) in 2003 found that mutant (KR33/35IS) in the context of H77c is not infectious in chimpanzee (23). Moreover, when alanine or glycine was substituted for histidine residue (H17), the virus infectivity was reduced by one log10 indicating that this residue, like H37 in M2, plays a role in enhancing virus production, whereas when histidine was replaced by alanine and glycine (the side chains of which are not protonated), a remarkable reduction was seen in the virus infectivity (78).
Recently, it has been demonstrated that p7 compound sensitivity varies according to viral genotype and using chimeric p7 proteins that neither the two TM helices nor the p7 basic loop individually determines compound sensitivity. Using point mutation analysis, from the three hydrophilic regions within the amino-terminal TM helix, only the conserved histidine at position 17 was found to be important for genotype 1b p7 channel activity and also identified a region at the p7 carboxy terminus, which may act as a specific sensitivity determinant for the drug amantadine. Mutations predicted to play a structural role affect both in channel function and oligomerization kinetics (81). The L(50–55)A mutant protein displayed essentially wild-type characteristics but showed differential sensitivity to amantadine and rimantadine. Logically, variation in hydrophobicity might be expected to affect drug binding, and some influenza amantadine escape variants have mutations affecting this region. Given that the polyleucine region is predicted to lie on the outside of the p7 channel, variation in this region may partially explain genotype-dependent drug sensitivity. Further mutagenesis studies are ongoing to map precise determinants of p7 inhibitor susceptibility (81).
Recently, it has been reported that p7 Sequence is not only important for its function but also effect other proteins functions. Confocal microscopy demonstrated that NS2 has a unique property of interacting with both structural and nonstructural proteins of HCV. Among mutation in p7, NS2 and Ns3 preventing infectious virus production, p7 mutant significantly reduced NS2 protein interactions by altering the membrane topology of the C-terminal domain of NS2 (82).
pH sensitivity and p7 activity
The HCV p7 regulates the internalization properties of HCV structural proteins (83). It has been hypothesized that deletion of p7 may lead to conformational changes of envelope proteins that are perhaps important for cell binding and entry of HCV virion as in the case of closely related viruses. A recent report found HCV E1 and E2 to be pH-sensitive and low pH rendered them incapable of permitting the entry of pseudotyped retroviral particles (84). Given the activity of p7 in the haemadsorption assay, it is speculated that one of its functions is to protect E1 and E2 from pH changes during the release of HCV particles from infected cells (31). As well as acting in membranes around mitochondria, the pool of p7 residing in the ER may also act in viral assembly. As an intracellular ion channel, p7 prevents pH gradient from developing inside the cells and causes a loss of acidity in multiple intracellular compartments, which is required for successful virus production (85). Moreover, the activity of the p7 ion channels is also pH dependent and the channels open at low pH (86), suggesting that the M2 and p7 proteins may share structural determinants as well, which are involved in encapsidation inside endosome during entry and un-coating the genome allowing the RNA replication (62, 85). Pseudotyped retroviruses presenting HCV E1 and E2 on their surface are known to show pH-dependent cell entry (84), likely because of E2 adopting a fusogenic conformation prematurely. It is conceivable that p7 may protect E2 from such pH-induced changes during assembly/entry in the same way that both it and M2 can protect influenza A virus haemagglutinin (31). The role of p7 in maintaining acidification has been demonstrated as when a sudden pH shift intracellular p7-containing vesicles equilibrate pH more rapidly than vesicles lacking p7 and this activity can be inhibited in a genotype-dependent manner (87). Similar with the case of other p7 activities, it is recently been reported that viral genome containing a p7KR mutant's inability to support infectious virus production and infectivity can be partially rescued by maintaining the pH gradient indicating that H+ gradient development is a necessary part in HCV life cycle fulfilled by the p7 protein (85).
Role of p7 in hepatitis C virus pathogenesis in virus/host interaction
Recently, it has become apparent that a number of virally expressed proteins interact with host genes. It is likely possible that HCV p7 is targeted to mitochondrial membranes in a similar fashion as other members of viroporins, which have the ability to affect apoptotic pathways via alteration of mitochondrial membrane permeability: e.g. HIV-1 Vpu (88), human T-cell lymphotropic virus-1 p13II (89), hepatitis B virus X protein (90, 91) and influenza virus PB1 ORF 2 (92), suggesting that it may modulate apoptosis. Moreover, p7 was found to be enriched in the heavy membrane fraction of 293 T-cell post-nuclear homogenates, which contain almost all the mitochondria as well as their associated ER-derived membranes. ER cisternae are known to wrap closely around mitochondria, facilitating rapid signalling and transport between the two organelles (93). In particular, the transmission of calcium ion fluxes from ER cisternae to mitochondria has been shown to be a pivotal process in the regulation of apoptotic signalling (94). It is tempting to speculate that the presence of p7 in these membranes may interfere with such signals, rendering the cell insensitive to pro-apoptotic stimuli from immune cells or the effects of other viral gene products.
Using the yeast two-hybrid system, Huang et al. (95) in 2005 showed that HCV p7 could bind to tetraspanins, the cell surface membrane proteins, indicating its role in HCV infection. Another important protein interacting with p7 protein from liver cDNA library was Homo sapiens nucleoporin 214 ku (NUP214) that serves for translocation of macromolecules between the nucleus and the cytoplasm along with other proteins such as immune responsive genes, signal sequence receptor, perinuclear RNA binding protein, etc. (95). The significance of proteins interaction with HCV p7 should be further studied in vivo and in vitro in detail for the analysis of importance of this region in HCV-induced pathogenesis.
Antiviral agents against p7 and hepatitis C virus therapy
Because of the limited antiviral effect and rapid emergence of viral resistance as a result of the current regime against HCV, the identification of new viral drug targets is especially important in the search for control strategies of the disease. New active anti-HCV agents against HCV polymerase and protease are under clinical development that will hopefully be available in the next decade, and more new targets are under studies. Theoretically, each step in viral life cycle and viral proteins can be a potential antiviral drug target. Taking together all the facts regarding p7 viroporin roles in HCV life cycle as an ion channel and late step virus replication cycle, it is predicted that drugs that presumably inhibit the virus production by blocking p7 activity directly or in combination may affect virus replication ultimately, resulting in reduced virus replication as a future antiviral strategy. Significantly, inhibition of the p7 ion channel activity was demonstrated with various long-alkyl-chain aminosugar derivatives (21, 96). Ion channels formed by p7 are cation selective at normal pH but can become less cation selective when solutions contain calcium ions. Hexamethylene amiloride, a drug previously shown to block ion channels formed by Vpu encoded by HIV-1, also blocked the HCV p7 activity (46).
Viroporin inhibitors were first approved 40 years ago in the form of antiinfuenza A drug amantadine and rimantadine establishing a successful pharmaceutical development for this clas of antiviral compounds. Amantadine and rimantadine inhibit influenza A by blocking H+ conductance through M2 ion channel affecting the conformational changes in the viral protein, which are essential for virus assembly (39, 97–99). Interestingly, the ion transport observed in lipid membranes because of HCV p7 protein could be blocked by a known ion channel inhibitor, Amantadine, which might have a beneficial effect in HCV treatment (20, 31, 100). Using chimera of HCV p7 and GBV-B p13 ion viroporins, it has been demonstrated that amantadine blocks the ion channel activity of both proteins in vitro and also replication of GBV-B in primary hepatocytes (101). Amantadine inhibits ion channel by preventing the protonation of a histidine residue and its subsequent interaction with the tryptophan residue in the channel lumen, thus preventing channel opening (37, 102–104). Interestingly, recent meta-analysis of amantadine has been used in clinical trials along with current regimes (IFN and Rib) and showed that this approach gave improved sustained vial responses in patients (100, 105–113). Contrary to this, a recent study of amantadine alongside IFN/Rib showed that, following an initial drop, virus load recovered after only a few weeks and this has also been observed for amantadine monotherapy (109, 114).
Comparison of p7 sequences reveals a high degree of variability, implying that p7 may differ in compound sensitivity in a sequence-specific fashion. In the light of this observation, Mihm et al. (115) in 2006 observed a substitution of L20F in nonresponders than responders with HCV-1b infection and combination therapy of IFN/Rib and amantadine, as this substitution changes the shape of the p7 ion channel pore and might impair amantadine action (115). Variation in p7 sequence, in particular those residues lining the hydrophilic face of the N-terminal α helix, which is thought to comprise the inner surface of the lumen (16, 54), could explain both the ineffectiveness of amantadine in clinical trials as well as the insensitivity of JFH-1HCV to the drug in culture (80). Griffin et al. (87) in 2008 confirmed multiple p7 inhibitor compounds using a parallel approach combining the HCV infectious culture system and a rapid throughput in vitro assay for p7 function and identified a genotype-dependent sensitivity of HCV to p7 inhibitors with separate sensitivity profiles for different p7 sequences, like amantadine, rimantadine, GSK2 and NNDNJ which primarily act by blocking the ion channel function of p7, whereas rimantadine blocked both virus release and p7 activity in vitro. Supporting the hypothesis that p7 sequence determines the binding of inhibitor compounds and may be p7 is a virion component (87). Chimeric proteins displayed intermediate drug sensitivities relative to the parental sequence indicating that residues conferring drug sensitivity must do so within the context of the overall channel structure. It is notable that considerable variation in pore-lining hydrophilic clusters within the amino-terminal helix exists between genotypes and often coincides with similar variation in the carboxy-terminal helix (81).
Amantadine perturbs the chemical shifts of a number of residues in the full-length protein; among the affected residues five are leucines in a TM helix (59). In a recent report, with these leucine residues are replaced by alanine residues, L(50–55)A, p7 exhibited greater resistance to the effects of amantadine (81). Preliminary results of mutagenesis and NMR spectroscopy are consistent in suggesting the involvement of the leucine-rich region of p7 in drug interactions (59). There is also evidence of involvement of the first TM helix residues, which include a histidine residue predicted on the basis of modelling to interact with amantadine (54).
Recently, a novel small molecule, BIT225 (N-[5-(1-methyl-1H-pyrazol-4-yl)-naphthalene-2-carbonyl]-guanidine), has been reported as having a potent stand-alone antiviral activity against HCV model pestivirus BVDV. BIT225 in combination with recombinant IFNα-2b showed synergistic effect alone and in addition with ribavin, and also with two nucleoside analogues (2′-C-methyl-adenosine and 2′-C-methyl-cytidine), which are know to inhibit the HCV-RNA-dependent RNA polymerase. The strong synergistic effect of BIT225, with recombinant IFN and Rib and anti-HCV nucleoside analogues represents the future development of BIT225 or related p7 inhibitors, for use in combination therapies for HCV treatment. Moreover, BIT225 has successfully completed phase 1a dose trial in healthy volunteers concerning single dose safety and pharmacokinetics, and in phase 1b of 7-day in selected doses in HCV-infected patients, representing a potential new class of antiviral compound against HCV (116).
Overall, these studies confirmed that the use of antiviral drugs against p7 has shown to significantly reduce the virus production. More studies are required to define how the primary sequence of p7 determines channel opening and drug sensitivity among different genotypes. Moreover, the use/development of p7 inhibitors for developing more potent compounds active against multiple p7 sequences either in combination with the current regime or alone will greatly aid in new future HCV therapeutic intervention to avoid/minimize the sequence-specific effect as all the above mentioned work has been able to reduce virus titre by 10-fold only at the concentrations used.
The HCV p7 is a small hydrophobic protein of 63 amino acids with two TM helical domains, TM1 and TM2, connected by a loop. P7 localizes primarily to the ER as an integral membrane protein and displays a topology where the amino- and carboxyl-terminal tails are oriented towards the ER lumen. p7 gene is located between the structural E2 and the nonstructural NS2 regions of the HCV polyprotein precursor. Cleavage of p7 is mediated by the ER signal peptidases of the host cell and locates in the cell ER. The role of p7 in the virus life cycle has been hard to define. The discovery that p7 displayed a cation-selective ion channel activity in vitro led to its classification within the ‘viroporin’ family that oligomerize to form channels that modify the permeability of the cell membrane to ions and other small molecule. It has been shown that p7 is not critical for RNA replication, although is critical for virus infectivity in vivo. Like the other members of viroporins family, HCV p7 is able to modify membrane permeability for efficient assembly and release of infectious virions in vitro and in vivo indicating that p7 is primarily involved in the late phase of the virus replication cycle. These properties make p7 an attractive candidate target for anti viral inter vention against hepatitis C infection. It is experimentally reported that targeting p7 in future antiviral therapies could potentially act by blocking HCV at multiple points in its life cycle using compounds like long alkylchain iminosugars derivatives, hexamethylene amiloride and amantadine as known ion channel blocker but the results of inhibitory effects in vitro and efficacy against infectious virus are controversial. Further experiments on in vitro functional analyses of ion channel function together with structural characterization are critical to define the precise function of p7 in HCV replication, pathogenesis and for the development of new anti viral drugs.