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)

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