1.A.53 The Hepatitis C Virus P7 Viroporin Cation-selective Channel (HCV-P7) Family
The mechanisms and functions of viral channel proteins have been reviewed by Fischer and Hsu (2011), Fischer et al. (2012) and Hyser and Estes 2015. The P7 (HCV-P7) viroporin of hepatitis C virus is of 63aas with 2 TMSs and is derived from the HCV polyprotein. P7 forms a heptameric ion channel with selectivity for monovalent cations and H+. Ion conduction has been demonstrated both in vivo (in cells) and in vitro (in lipid bilayers) (Clarke et al., 2006). Its ion channel activity is inhibited by amantadine and rimantadine, long alkyl chain imino-sugar derivatives, and amiloride. Another peptide derived from the HCV polyprotein, NS4A, (YP_001491554; 54aas) has also been reported to form pores (Madan et al., 2007). The structure and function of the p7 protein has been reviewed (Atoom et al. 2014). Retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at the p7 amino-terminus (Denolly et al. 2017). A broad consensus among the p7 variants indicates that distantly related HCVs preserve key features of structure and dynamics (Oestringer et al. 2019).
The two transmembrane domains of p7 are separated by a short basic loop. It forms a cation selective channel whose activity in suspended lipid bilayers. The p7 protein is essential for HCV replication, assembly, release and production of infectious progeny virions (Khaliq et al., 2011). It is required for production of infectious virus in cell culture for the related bovine viral diarrhea virus. It can transport protons, other cations, and carboxyfluorescein (Gan et al. 2014). TMS1 in an α-helical configuration faces the lumen of the channel. There seem to be two forms, one that transports both H+ and carboxyfluorescein, and one that transports H+ but not carboxyfluorescein (Gan et al. 2014). Inhibitors of p7 have been identified (Mathew et al. 2015).
P7 belongs to a functional family of viral ion channels known as viroporins. These are small integral membrane proteins that homo-oligomerize to form channels that mediate cation fluxes across cellular and viral membranes. Precedent for these proteins as therapeutic targets in a clinical setting is provided by the M2 protein of influenza A virus (TC #1.A.19), which is also sensitive to amantadine and related derivatives. p7 is able to replace M2 in a functional assay. Amantadine inhibited both M2 and p7 in this assay at the same concentrations. The basic loop in the cytoplasm is critical for p7 function (Khaliq et al., 2011).
The non-structural protein p7 of Classical Swine Fever Virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa (70 aas; derived from polyprotein PolG_CSFVA (P19712), 3898 aas). The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that is a cytosolic loop. CSFV p7 is critical for virus production and virulence (Gladue et al. 2012). Structure-function analyses in model membranes emulating the ER lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine. The PolG_CSFVA polyprotein shows low sequence similarity with polyproteins in 1.G.3. CSFVA viroporin is homologous to viroporins from Border disease virus, Bovine diarrhea virus 1, Pronghorn antelope pestivirus and Pestivirus giraffe-1.
As noted above, the p7 peptide contains two putative transmembrane regions connected by a short hydrophilic segment. Expression of p7 protein in E. coli leads to permeabilization of bacterial cells to small molecules (Guo et al., 2013). p7 also enhances the permeability of mammalian cells, increasing the intracellular Ca2+ concentration and the permeability of cells to the translation inhibitor Hygromycin B. It forms homo-oligomers and localizes to the ER at the early stage of expression. It can be transferred to the plasma membrane at a later stage. Detergent permeabilization assays confirmed that p7 is a 2-TMS protein with its N- and C-termini exposed to the ER lumen. Deletion analysis suggested that amino acyl residues 41-63 are essential for viroporin activity of the protein (Guo et al., 2013).
GB virus B (GBV-B), a hepatotropic virus, is closely related to hepatitis C. It infects small New World primates and replicates efficiently in primary hepatocyte cultures and is an attractive surrogate model system. Ghibaudo et al. 2004 characterized signal peptidase processing of the polyprotein segment containing the putative structural proteins. They identified the exact N termini of the mature GBV-B envelope proteins, E1 and E2, and the first nonstructural protein, NS2, by direct amino acid sequencing. These studies document the existence of a previously unrecognized 13-kDa protein (p13) located between E2 and NS2 within the polyprotein. The p13 protein sequence was compared to that of hepatitis C virus p7, a small 2 TMS membrane-spanning protein with a similar location in the polyprotein that has ion channel activity. The C-terminal half of p13 is homologous to p7, suggesting a common function, but the substantially larger size of p13, with 4 rather than 2 predicted transmembrane segments, indicates a different structural organization and/or additional functions. The identification of p13 in the GBV-B polyprotein provides support for the hypothesis that ion channel-forming proteins are essential for the life cycle of flaviviruses, possibly playing a role in virion morphogenesis and/or virus entry into cells (Ghibaudo et al. 2004).
The p7 viroporin of the hepatitis C virus (HCV) forms an intracellular proton-conducting transmembrane channel in virus-infected cells, shunting the pH of intracellular compartments, and thus, helping virus assembly and release. The protein sequences and drug sensitivities of p7 proteins vary between the seven major genotypes of the hepatitis C virus, but the essential channel activity is preserved. Breitinger et al. 2021 investigated the effect of several inhibitors on recombinant HCV p7 channels corresponding to genotypes 1a-b, 2a-b, 3a and 4a. They established a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assay for recombinant p7 expressed in HEK293 cells to assess channel activity. Hexamethylene amiloride (HMA) was the most potent inhibitor but possessed cytotoxic activity at higher concentrations. Rimantadine was active against p7 of all genotypes, while amantadine activity was genotype-dependent. Thus, cell viability assays can be used to assess viroporin activity and identify channel inhibitors.
The reaction catalyzed by the heptameric p7 pore is:
Cations (in) cations (out)
Hepatitis C Virus, HCV-P7, of 63 aas and 2 TMSs (Clarke et al., 2006). It's mechanism and function have been investigated in considerable detail (Gan et al. 2014). Histidine-17, which faces the lumen of the pore when protonated, allows Cl- entry, but deprotonation also allows Ca2+ entry. Imposition of voltage creates a Cl- current (Wang et al. 2014). The structure and dual pore/ion channel activity of p7 of different HCV genotypes have been reviewed (Madan and Bartenschlager 2015). It may transport protons (Scott and Griffin 2015); it's structure has been determined by NMR (Montserret et al. 2010) and by electron microscopy (Luik et al. 2009). The p7 N-terminal helical region is critical for E2/p7 processing, protein-protein interactions, ion channel activity, and infectious HCV production (Scull et al. 2015). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor. The retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway (Denolly et al. 2017). Chen et al. 2018 provided evidence that the oligomeric channel is a cation-selective hexamer. The his-9 in the hexameric model forms a first gate, acting as a selectivity filter for cations. while valines form a second gate, serving as a hydrophobic filter for dehydrated cations. The binding pocket for the channel blockers, amantadine and rimantadine, is composed of residues 20-26 in H2 helix and 52-60 in H3 helix (Ying et al. 2018). Two of the best inhibitors were ARD87 and ARD112 (Wei et al. 2021).
P7 of hepatitis C virus (63 aas; 2 TMSs; CAH23613)
Channel forming Hepatitis C virus NS4a peptide (54 aas) (viroportin NS4a). The NS4a peptide has been shown to form pores (Madan et al. 2007). This protein is a peptide fragment of the large glycoproteins of the Hepatitis C Virus.
Hepatitis C virus NS4a peptide (D2K2A7)
Hepatitis GB virus B (GBV-B) polyprotein of 2864 aas, containing the p13 viroporin. It is found between residues 614 and 733 in the polyprotein and has 4 predicted TMSs. It is homologous to but larger than the p7 protein of hepatitis C virus (Ghibaudo et al. 2004).
Polyprotein B of GBV-B virus
p7 protein from the hepatitis C virus subtype 3a polyprotein. Molecular interactions between NS2 and p7 and E2 have been observed, and the NS2 transmembrane region is required for both E2 interaction and subcellular localization. Specific mutations in core, envelope proteins, p7 and NS5A abolish viral assembly (Popescu et al. 2011).
p7 of hepatitis C virus subtype 3a
Polyprotein containing p7 of hepacivirus AK. Ion channel activity has been demonstrated in lipid bilayers (Walter et al. 2016).
Polyprotein of hepacivirus AK
Classical Swine Fever Virus (CSFVA) p7 viroporin (70 aas; 2 TMSs) which probably transports Ca2+ and other inorganic cations (Scott and Griffin 2015). The p7 protein induces IL-1β secretion which is inhibited by the ion channel blocker amantadine. The p7 protein is a short-lived protein degraded by the proteasome (Lin et al. 2014). CSFV-p7 forms pores wide enough to allow ANTS (MW, 427 Da) release. Two pore structures with slightly different sizes and opposite ion selectivities were detected (Largo et al. 2016). The relative abundances of these pore types depend on membrane composition suggesting that the physicochemical properties of the lipid bilayers present in the cell endomembrane system modulate viroporin activity. Permeabilization of ER membranes by CSFV p7 depends on two sequence determinants: the C-terminal transmembrane helix (involved in pore formation), and the preceding polar loop that regulates its insertion and activity. The pore-forming domain of p7 may assemble into finite pores with approximate diameters of 1 and 5nm. Formation of the larger pores can hamper virus production without affecting ER localization or homo-oligomerization (Largo et al. 2018). p7 specifically interacts with host protein CAMLG, an integral ER transmembrane protein involved in intracellular calcium release regulation and signal response generation. Mutants of p7 have decreased virulence in swine (Gladue et al. 2018).
CSFVA P7 viroporin of Classical Swine Fever Virus (Q9YS30)
The borine viral diarrhea virus (BVDV) p7 peptide, viral budding process initiator. It probably transports H+ and other cations (Scott and Griffin 2015).
p7 of Bovine viral diarrhea virus (AAB47140) polyprotein: Q96662.
Genome polyprotein [Cleaved into: Core protein p21 (Capsid protein C) (p21); Core protein p19; Envelope glycoprotein E1 (gp32) (gp35); Envelope glycoprotein E2 (NS1) (gp68) (gp70); p7; Protease NS2-3 (p23) (EC 3.4.22.-); Serine protease NS3 (EC 188.8.131.52) (EC 184.108.40.206) (EC 220.127.116.11) (Hepacivirin) (NS3P) (p70); Non-structural protein 4A (NS4A) (p8); Non-structural protein 4B (NS4B) (p27); Non-structural protein 5A (NS5A) (p56); RNA-directed RNA polymerase (EC 18.104.22.168) (NS5B) (p68)]. The core protein (C) corresponds to residues 1 - 233 in the polyprotein (GenBank acc. # AFS60319.1) (Devi et al. 2022). Proteolytic intermediates of C with an intact transmembrane ER-anchor assemble into pore-like structures in the ER membrane (Devi et al. 2022).
POLG_HCVVN of Hepatitis C virus genotype 6d
Hepatitis C virus p7 protein. The NMR structure is available. The channel is cation-selective and is inhibited by hexamethylene amiloride but not by amantadine. The protein has an N-terminal α-helix that precedes TMS1, and TMSs 1 and 2 are connected by a long cytosolic loop bearing a dibasic motif (Montserret et al. 2010). p7 forms an ion channel and is indispensable for HCV particle production. Although the main target of HCV p7 is the endoplasmic reticulum, it also targets mitochondria., causes mitochondrial depolarization and ATP depletion, and causes mitochondrial dysfunction to support HCV particle production (You et al. 2017).
p7 of Hepatitis C virus strain HCV-J (genotype 1b)
Viroporin of 63 aas and 2 TMSs.
Viroporin of Bovine viral diarrhea virus (BVDV) (Mucosal disease virus)