Intracellular Proton Conductance of the Hepatitis C Virus p7 Protein and Its Contribution to Infectious Virus Production

The hepatitis C virus (HCV) p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H+) conductance in vesicles and was able to rapidly equilibrate H+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5) vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection, possibly through protecting nascent virus particles during an as yet uncharacterized maturation process

NMR studies of p7 protein from hepatitis C virus

The p7 protein of hepatitis C virus (HCV) plays an important role in the viral lifecycle. Like other members of the viroporin family of small membrane proteins, the amino acid sequence of p7 is largely conserved over the entire range of genotypes, and it forms ion channels that can be blocked by a number of established channel-blocking compounds. Its characteristics as a membrane protein make it difficult to study by most structural techniques, since it requires the presence of lipids to fold and function properly. Purified p7 can be incorporated into phospholipid bilayers and micelles. Initial solid-state nuclear magnetic resonance (NMR) studies of p7 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 transmembrane domain 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. The addition of the channel blocker amantadine to the full-length protein resulted in selective chemical shift changes, demonstrating that NMR has a potential role in the development of drugs targeted to p7

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

Infection with the hepatitis C virus (HCV) has a huge impact on global health putting more than 170 million people at risk of developing severe liver disease. The HCV encoded p7 ion channel is essential for the production of infectious viruses. Despite a growing body of functional data, little is known about the 3-dimensional (3D) structure of the channel. Here, we present the 3D structure of a full-length viroporin, the detergent-solubilized hexameric 42 kDa form of the HCV p7 ion channel, as determined by single-particle electron microscopy using the random conical tilting approach. The reconstruction of such a small protein complex was made possible by a combination of high-contrast staining, the symmetry, and the distinct structural features of the channel. The orientation of the p7 monomers within the density was established using immunolabeling with N and C termini specific Fab fragments. The density map at a resolution of ≈16 Å reveals a flower-shaped protein architecture with protruding petals oriented toward the ER lumen. This broadest part of the channel presents a comparatively large surface area providing potential interaction sites for cellular and virally encoded ER resident proteins

Direct visualization of the small hydrophobic protein of human respiratory syncytial virus reveals the structural basis for membrane permeability

AbstractHuman respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract disease in infants. The HRSV small hydrophobic (SH) protein plays an important role in HRSV pathogenesis, although its mode of action is unclear. Analysis of the ability of SH protein to induce membrane permeability and form homo-oligomers suggests it acts as a viroporin. For the first time, we directly observed functional SH protein using electron microscopy, which revealed SH forms multimeric ring-like objects with a prominent central stained region. Based on current and existing functional data, we propose this region represents the channel that mediates membrane permeability.Structured summaryMINT-7890792, MINT-7890805: SH (uniprotkb:P04852) and SH (uniprotkb:P04852) bind (MI:0407) by chromatography technology (MI:0091)MINT-7890784, MINT-7890776: SH (uniprotkb:P04852) and SH (uniprotkb:P04852) bind (MI:0407) by electron microscopy (MI:0040