Determination of Pore-Lining Residues in the Hepatitis C Virus p7 Protein

Abstract

The p7 protein from hepatitis C virus is critical for the assembly and secretion of infectious virus, making it an attractive drug target. It is thought to be a viroporin with a demonstrated ion channel activity when reconstituted into planar lipid bilayers. Electron microscopy experiments suggest that p7 oligomers coexist as hexamers and heptamers. Proposed models of p7 oligomers assume the N-terminal helix to be the pore lining helix. Here, we demonstrate, via electrophysiology, that Cu(2+) has an inhibitory effect on the p7 ion channel and that the amino acid responsible for this inhibition is one histidine in each monomer. This information coupled with the p7 sequence data suggests that the N-terminal helix of p7 does indeed form the transmembrane pore and that this histidine is pore-lining. The information will aid in the construction of oligomeric pore-models and the interpretation of electron microscopy data.

The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy

“…HCV p7 (strain JFH-1, genotype 2a) was chemically synthesized and purified to Ͼ95% by reverse phase high-performance liquid chromatography. The resulting peptides formed active ion channels in artificial membranes (15). For the current study, we dissolved p7 peptides in the mild detergent 1,2-diheptanoyl-snglycero-3-phosphocholine (DHPC) and determined their oligomerization state by blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows proteins to maintain their undenatured native state and oligomerization pattern.…”

“…p7 belongs to the viroporins, small virally encoded proteins with at least 1 membrane-spanning helix that oligomerize to form channels or pores that modify the permeability of the cell membrane to ions and other small molecules (7). In planar lipid bilayers, p7 monomers oligomerize to form cation-selective ion channels that can be specifically inhibited by long alkylchain iminosugars, amiloride, and amantadine derivatives, with varying reported efficacies (6,(8)(9)(10)(11)(12)(13)(14)(15). Each HCV p7 monomer consists of 63 aa, most of which are hydrophobic and possibly contain endoplasmic reticulum (ER) retention signals (16-18).…”

“…The loop is assumed to face the cytoplasm, with the N and C termini facing the ER lumen. Recent electrophysiological experiments suggest that the Nterminal helix lines the pore (15). Electron microscopy studies aimed at defining the oligomerization state of the p7 channel have suggested that monomers assemble into either hexamers (10) or heptamers (9) in lipid bilayers.…”

Viroporins: structure, function and potential as antiviral targets

“…At one end of the spectrum, more 'channel-like' viroporins such as M2 display discrete single-channel events in artificial bilayers, reminiscent of cellular ion channels (Duff & Ashley, 1992). Further along the spectrum towards a pore, HCV p7 has been shown to adopt both single-channel and 'burst-activity' behaviour in bilayers, with single-channel activity also comprising more than one conductance state (Chew et al, 2009;Clarke et al, 2006;Griffin et al, 2003;Pavlović et al, 2003;Premkumar et al, 2004;Whitfield et al, 2011). This may reflect p7 behaviour whilst conducting a non-preferred ionic substrate, although p7 activity is also differentially modulated by several factors, including virus genotype (Atkins et al, 2014), the formation of different oligomers (Clarke et al, 2006;Luik et al, 2009) and membrane composition (Whitfield et al, 2011), all of which may influence the meta-stable nature of channel complexes (Chandler et al, 2012).…”

“…These include a role for positions 17 and 21, occupied by His and Tyr/Trp in many, but certainly not all HCV isolates, as an M2-like HxxxW proton sensor/gate motif (Meshkat et al, 2009). However, genotype 1a (H77) channels retain His17 and are not pH-activated (Atkins et al, 2014;Chew et al, 2009;Li et al, 2012). Ser/Tyr21, Trp30 and Tyr/His31 have also been shown to modulate channel activity and/or infectious virion production in various studies (Brohm et al, 2009;Steinmann et al, 2007a;StGelais et al, 2009).…”