Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel

The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target

Two different conformations in hepatitis C virus p7 protein account for proton transport and dye release

The p7 protein from the hepatitis C virus (HCV) is a 63 amino acid long polypeptide that is essential for replication, and is involved in protein trafficking and proton transport. Therefore, p7 is a possible target for antivirals. The consensus model for the channel formed by p7 protein is a hexameric or heptameric oligomer of α-helical hairpin monomers, each having two transmembrane domains, TM1 and TM2, where the N-terminal TM1 would face the lumen of this channel. A reported high-throughput functional assay to search for p7 channel inhibitors is based on carboxyfluorescein (CF) release from liposomes after p7 addition. However, the rationale for the dual ability of p7 to serve as an ion or proton channel in the infected cell, and to permeabilize membranes to large molecules like CF is not clear. We have recreated both activities in vitro, examining the conformation present in these assays using infrared spectroscopy. Our results indicate that an α-helical form of p7, which can transport protons, is not able to elicit CF release. In contrast, membrane permeabilization to CF is observed when p7 contains a high percentage of β-structure, or when using a C-terminal fragment of p7, encompassing TM2. We propose that the reported inhibitory effect of some small compounds, e.g., rimantadine, on both CF release and proton transport can be explained via binding to the membrane-inserted C-terminal half of p7, increasing its rigidity, in a similar way to the influenza A M2-rimantadine interaction.Published versio

Incorporation of p7 into lipid bilayers.

<p>(A) Schematic representation of the flotation system, where fractions with increasing density are found: (LIP), fraction with expected liposome fraction and (PEL), the most dense fraction; (B) SDS-PAGE gel corresponding to samples in the addition experiment: (BF) sample after addition and before liposome flotation, (PEL) sample at the bottom of the tube (see A), (LIP) sample associated to liposomes (see A). Sample loading was nominally 4 µg (1×) except in samples PEL and LIP, as indicated. Arrows and dots are shown for reference to indicate significant bands; (C) same as (B), but for the extruded sample where detergent was removed before extrusion; (D) ATR-IR spectra of the LIP fractions for the ‘addition’ sample (red) and the ‘extrusion’ sample (blue). Spectra for other p7 containing fractions were similar and are not shown; (E) same as (D), after a mild deconvolution (FWHH = 25 cm⁻¹ and k = 1.5). The main maxima in the amide I region are indicated.</p

Secondary structure of p7 protein in lipid bilayers.

<p>(A) amide I region corresponding to recombinant p7 protein reconstituted into hydrated DMPC bilayers by the dialysis method, after having been solubilized and dried from methanol (M, blue) or HFIP (H, red); (B) Same for a sample reconstituted by the direct method after drying from methanol (1), after freeze-thawing (2), after extrusion (3), and supernatant after 5 min centrifugation of the proteoliposomes at 2,000 g (4); (C) Same for sample obtained by the addition method, where p7 was dissolved at ∼10 mg/ml in the indicated solvents: MeOH, HFIP or TFE; (D), Amide I region of dry recombinant p7 protein obtained after lyophilizing a solution of p7 dissolved in either methanol (M) or HFIP (H).</p

Expression and purification of coronavirus envelope proteins using a modified β-barrel construct

Coronavirus envelope (E) proteins are short (∼100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2–3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides

Secondary structure of N- and C-terminal fragments of p7.

<p>Infrared amide I region of (A) fragment p7(1-26), and its Fourier self deconvolved spectrum (B); C and D, same for fragment p7(27-63). Samples pre-solubilized and dried from methanol and HFIP are indicated as dotted and solid lines, respectively.</p

Overexpression and purification of p7 protein from <i>E. coli</i>.

<p>(A) Amino acid sequence of full-length p7, where extra residues SNAM (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078494#s2" target="_blank">Materials and Methods</a>) are present in the recombinant form and extra M in the synthetic form; (B) A band migrating near 50 kDa was observed after IPTG induction which corresponds to MBP-p7 fusion protein (arrow). BI, before IPTG induction; AI, 16 hours after IPTG induction; (C) Ni²⁺-NTA purification of MBP-p7 with close to 85% purity after elution in 500 mM imidazole. Ly, supernatant of total cell lysate; FT, flow through from Ni²⁺-NTA column; E, eluent from Ni²⁺-NTA column (D) TEV digestion results of MBP-p7 at room temperature, at time 0 and after 4 h incubation with gentle shaking; (E) RP-HPLC purification of p7 with a C3 RP-HPLC chromatography column. The peak corresponding to purified p7 is indicated by an arrow.</p

Proton transport of p7 and its fragments.

<p>Comparison of liposomal proton transport mediated by p7 full length, p7-TM1 fragment and p7-TM2 fragment solubilized in HFIP prior mixing with lipids (protein to lipid molar ratio 1∶125); LIP, negative control without peptide.</p

Liposome based proton transport assay.

<p>(A) Proton transport mediated by recombinant p7 pre-solubilized in HFIP at the p7/lipid 1∶125 molar ratio. Three different lipid mixtures were used for the assay, <i>E. coli</i> lipids, PA/PE/PS/PC (PAESC), 5∶2∶2∶1 (w/w) and PA/PE/PC/Cholesterol (PACC), 5∶2∶2∶1 (w/w). PACC and PAESC represent negative control, i.e., liposomes without p7; (B) Proton transport mediated by recombinant p7, where p7 was solubilized in HFIP (H) or methanol (M) and mixed with PAESC lipids at protein to lipid ratios indicated. For comparison, a trace using M2(18-60) in <i>E. coli</i> lipids in also shown at the protein to lipid molar ratio indicated; (C) Same as above, showing the effect of 5 µM rimantadine (+Rim). PAESC, negative control, i.e., liposome without p7.</p

Schematic view of the proposed two forms of p7 (A and B).

<p>(1) In form B, TM2 is embedded in the lipid bilayer and the extra β-structure is contributed by exposure of TM1 to the aqueous environment. This form is able to release CF (black arrow), but not protons (grey arrow). As part of the B form extends into the extramembrane domain, it is likely to participate in fusion events or membrane destabilization; (2) Form A has two TM domains, TM1 and TM2, separated by a loop, where TM2 lines the lumen of the channel. This form is unable to release CF, but it is able to transport protons.</p