key: cord-0836126-399rq84t
authors: Wilson, Lauren; Gage, Peter; Ewart, Gary
title: Validation of Coronavirus E Proteins Ion Channels as Targets for Antiviral Drugs
date: 2006
journal: The Nidoviruses
DOI: 10.1007/978-0-387-33012-9_104
sha: a47a4d626d0cac56efd79a46eb6749d6f7cd4c6f
doc_id: 836126
cord_uid: 399rq84t

nan

Coronaviruses are divided into three groups, depending on the sequence homology and antigen cross-reactivity. Groups 1 and 2 contain the mammalian coronaviruses, and group 3 consists of the avian coronaviruses. All coronavirus groups encode E protein, a small, 9-12 kDa integral membrane protein. 1 Although, there is little sequence homology between the coronavirus groups, all E proteins share structural homology, they all contain an N-terminus, which consists of a short 7-9 amino acid hydrophilic region, and a 21-29 amino acid hydrophobic transmembrane domain, followed by a hydrophilic C-terminal region. 2 The exact functions and mechanisms of the coronavirus E proteins are yet to be established, although E proteins have been shown to be important for coronavirus replication, mediating viral assembly, and morphogenesis.

Coronavirus E proteins share several characteristics with viral ion channels, which are small hydrophobic virus-encoded proteins. Virus ion channels have a highly hydrophobic domain that forms at least one amphipathic α-helix that oligomerizes to form an ion-conductive pore in membranes. Virus ion channels function to modify the cells permeability to ions and have been shown to mediate viral entry/exit or virus assembly and budding. 3, 4 The first identified viral ion channel, hence the best characterized, is the M2 protein encoded by influenza A. M2 forms proton selective ion channels that mediates viral uncoating and protects acid-sensitive hemagglutinin glycoprotein during transport to the cell surface. 5 Although influenza B does not encode the M2 ion channel, it has been demonstrated to have two ion channel forming proteins, NB and BM2, 5,6 whose role in viral replication are currently being investigated. Since the identification of the M2 ion channel, several other viruses have been demonstrated to encode viral ion channels. The HIV-1 accessory protein Vpu has also been shown to have ion channel activity, which mediates the release of viral particles from the plasma membrane. 6, 7 Most recently, the 6K proteins from the alphaviruses, Ross River Virus and Barmah Forest Virus, have been shown to form cation-selective ion channels in planar lipid bilayers. 6 Additionally, several authors have shown ion channel activity of the hepatitis C virus (HCV) p7 protein. [8] [9] [10] The M2 channel is well established as a target for antiviral drug therapy, and blockers of other viral ion channels have been shown to inhibit replication of the parent virus. The M2 ion channel is inhibited by amantadine and some of its derivates, which are currently used as clinical treatment of influenza A infection. 11, 12 The Vpu ion channel activity in planar lipid bilayers is inhibited by the amiloride derivatives 5-(N,Nhexamethylene)amiloride (HMA) and 5-(N,N-dimethyl)amiloride (DMA), but not by amiloride itself. Furthermore, HMA inhibits Vpu enhancement of Gag-driven virus-like particles (VLP) budding from HeLa cells co-expressing Vpu and Gag and also inhibits HIV-1 replication in cultured primary human macrophages. 6 In addition, the p7 ion channel has been shown to be inhibited by HMA, amantadine, and long-alkyl chain immunosugar derivatives. [8] [9] [10] Thus, a number of precedents have been set for ion channels as targets of potential antiviral compounds.

Due to coronavirus E proteins similarities with viral ion channels and their important role in viral replication, we hypothesized that coronavirus E proteins have ion channel activity, and compounds that block these channels may result in inhibition of viral replication. We report here that representative E proteins from all three coronavirus groups form ion channels. Furthermore, we found that certain amiloride derivates block E protein ion channel activity and inhibit replication of coronaviruses in cultured cells.

E peptides corresponding to the human coronavirus 229E (HCoV-229E) E protein (MFLKLVDDHALVVNVLLWCVVLIVILLVCITIIKLIKLCFTCHMFCNRTVYGPIK NVYHIYQSYMHIDPFPKRVIDF), GenBank accession number NP_073554, mouse hepatitis virus (MHV)-A59 E protein (MFNLFLTDTVWYVGQIIFIFAVCLMVT IIVVAFLASIKLCIQLCGLCNTLVLSPSIYLYDRSKQLYKYYNEEMRLPLLEVDDI), GenBank accession number NP_068673, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) full-length E protein (MYSFVSEETGTLIVNSVLLFLAFVVF LLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV), SARS -CoV N-terminal E protein (MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAIL TALRLC), GenBank accession number NC004718, and infectious bronchitis virus (IBV) Beaudette strain E protein (MTNLLNKSLDENGSFLTALYIFVGFLALYLLGRA LQAFVQAADACCLFWYTWVVVPGAKGTAFVYNHTYGKKLNKPELETVINEFPK NGWKQ), GenBank accession number CAC39303, were chemically synthesized and purified, as described previously. 13, 14 The peptides were shown to contain full-length products by a variety of methods including, Western blot analysis with E protein specific antibodies, and mass spectral analysis. 13,14

The HCoV-229E, MHV, SARS-CoV, and IBV purified E proteins were resuspended to 1 mg/ml in 2,2,2-trifluoroethanol (TFE) and their ability to form ion channels was tested on a Warner bilayer rig (Warner instruments, Inc. Hamden, CT), as described previously. 13,14

A 50 mM stock solution of compounds was prepared in 50% DMSO, 50% methanol, which was further diluted for use in ion channel inhibition studies and in the antiviral assays.

To determine if the amiloride derivatives blocked SARS-CoV E protein ion channel conductance in planar lipid bilayers, after ion channel current amplitude was detected, 100-200 µM of compound was added to the CIS chamber while stirring. The current across the bilayer was recorded prior to addition of SARS-CoV E protein, after detection of ion channel conductance, and after addition of the compound. T-test (Microsoft Excel) was used to test the difference between the normalized mean currents before and after addition of the compound.

The virus plaque phenotype in the presence or absence of antiviral compound was studied in L2 cells (ATCC). The L2 cells were plated in 6-well plates and grown to confluence, then infected with a MOI 0.01 of MHV-A59 (ATCC) or MOI 0.1 of MHV∆E or MHVSARS E (kind gift from Paul Masters, Wadsworth Center, Albany, NY) for 1 hour. The virus was removed and replaced with 1% seaplaque overlay in MEM supplemented with 10% FCS and 20 µM or 0 µM of antiviral compound in 50% DMSO 50% methanol. After 48 hours incubation at 37°C/5% CO 2, the cells were stained with 0.1% crystal violet in 20% methanol.

To determine the Selectivity Index (SI) of the antiviral compounds the effective concentration 50 (EC 50 ) was calculated by plaque assay on L929 cells (ATCC) and the toxicity concentration 50 (TC 50 ) was calculated by MTT cytoxicity assay. The SI was calculated by dividing the TC 50 by the EC 50 50 /EC 50 ).

HCoV-229E, MHV, IBV, SARS-CoV -full-length and -N-terminal E proteins were tested for their ability to form ion channels in planar lipid bilayers. Experiments were done to determine E proteins ion selectivity for Na + over Cland K + over Clions. Figure   had divergent ion channel selectivity. The group 1 coronavirus HCoV-229E E protein is K + selective, whereas the group 2 MHV and SARS-CoV E proteins, as well as the group 3 coronaviruses, IBV E protein were more selective for Na + ions. The different E protein ion channel selectivity may reflect subtly divergent roles of the E protein groups in the coronavirus life cycle, although this remains to be established. In support of this idea is 575 (SI = TC 1 shows typical ion channel conductance of representatives from coronavirus E proteins from group 1, 2, or 3, demonstrating that E protein ion channel activity is a general property of all coronavirus groups. Interestingly, the E proteins from the different coronavirus group 

Because it is possible that E protein ion channel activity is important for coronavirus replication and a precedent has been set that ion channels are suitable targets of antiviral therapy, we tested amiloride, plus, its derivatives HMA, and 5-(N-Methyl-N-576 that the group 1 -K + selective -E proteins are essential for viral replication, 15 whereas group 2 (Na + selective) E proteins are important, but not essential for coronavirus replication. 16 Further data to support this theory was presented at this conference (see Refs. 17 in this proceedings book), Masters et al., 2006 demonstrated that the Na + group 3 IBV could substitute for the Na + selective MHV E protein and enhance replication of recombinant MHV virus. On the contrary, the group 1 transmissible gastroenteritis virus E protein, which our data suggest may be K + selective, could not substitute for the MHV E protein in recombinant viruses. 17, 18 Because, the group 1 and 2 coronavirus E proteins share more sequence homology than the group 2 and 3 E proteins, 18, 19 the ability of the different E protein groups to substitute for the MHV E protein could be more dependent on their ion channel selectivity than the sequence homology.

Previously, it has been demonstrated that the transmembrane (TM) domain of several ion channels, including M2 and Vpu, form channels with similar properties as the fulllength proteins. 18, 19 Therefore, we tested the ability of the SARS-CoV E protein Nterminal first 40 amino acids, which encompass the putative TM domain for its ability to form ion channels in planar lipid bilayers. Indeed, the SARS-CoV N-terminal peptide formed ion channels that had similar properties to the full-length E protein. Thus, the hydrophilic C-terminal domain is dispensable for ion channel activity in planar lipid bilayers. 13 Several studies are currently being conducted to determine if E proteins TM domain ion channel activity is important for coronavirus replication (see Refs. 20 and 21 in this proceedings book). Intriguingly, these studies have shown that substitution or mutation of the E protein TM domain is detrimental for viral replication, suggesting that E protein ion channel activity could be important for coronavirus replication. selective E proteins from group 2, bovine coronavirus and SARS-CoV, as well as the isobutyl)amiloride (MIA) for their ability to inhibit SARS-CoV E protein ion channel conductance. Table 1 demonstrates that HMA reduced the conductance across the bilayer by about 80%, MIA by about 70%, while amiloride itself did not significantly reduce the current across the bilayer (Table 1 ).

We found that HMA and MIA inhibited the SARS-CoV E protein ion channel conductance in planar lipid bilayers; therefore, we wanted to test their ability to inhibit coronavirus replication. Due to the safety issues and difficulties of working with the SARS-CoV, we decided to use the recombinant MHV virus that expresses the SARS-CoV E protein in place of the MHV E protein (MHVSARS E). The MHVSARS E virus replicates efficiently in cultured mouse cells, but has a slightly smaller plaque phenotype than the wild-type MHV.

has a small plaque phenotype. MHV wild-type virus has a plaque phenotype of about 3-4 mm in the absence of antiviral compound, but in the presence of 20 µM HMA or MIA the plaque size is reduced to about 1 mm. Similarly, 20 µM of HMA or MIA reduced the MHVSARS E plaque size from about 2-3 mm to 1 mm. In contrast, neither HMA nor MIA significantly affected MHV∆E plaque phenotype. Comparable with the ion channel conductance study, amiloride did not have any significant effect on MHV, MHVSARS E or MHV∆E replication ( Table 1 ). The selectivity index (SI) of HMA and MIA on MHV and MHVSARS E replication were greater than 10, indicating that the antiviral activity of the compounds are notably removed from toxicity (Table 1) . Further, the antiviral activity of these compounds against MHV expressing the homo-or heterologous E proteins, but not against the ∆E construct, together with direct mechanism of action of the compounds is via inhibition of E protein ion channel activity. observations of SARS-CoV E channel blockage, is supportive of our hypothesis that the the entire E protein deleted (MHV∆E), which replicates to low titre in mouse cells and

As a control we also used the MHV recombinant virus with

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Small envelope protein E of SARS: Cloning, expression, purification, CD determination, and bioinformatics analysis

Viral ion channels: Structure and function

Viral Membrane Proteins: Structure, Function, and Drug Design

Viral Membrane Proteins: Structure, Function, and Drug Design

The two biological activities of human immunodeficiency virus type 1 vpu protein involve two separable structural domains

Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride

The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, amantadine

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkylchain iminosugar derivatives

Influenza virus M2 protein has ion channel activity

Managing influenza: Amantadine, rimantadine and beyond

SARS coronavirus E protein forms cation-selective ion channels

coronavirus replication (submitted)

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Human immunodeficiency virus type 1 vpu protein affects Sindbis virus glycoprotein processing and enhances membrane permeabilization

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Identification of an ion channel activity of the vpu transmembrane domain and its involvement in the regulation of virus release from HIV-1-infected cells

The transmembrane domain of influenza a M2 protein forms amantadinesensitive proton channels in planar lipid bilayers

The small envelope protein E is not essential for murine coronavirus replication

Role of the mouse hepatitis coronavirus envelope protein transmembrane domain

Hexamethylene amiloride blocks E protein ion channels and inhibits