key: cord-0993445-lfqm3iuw
authors: Breitinger, Ulrike; Farag, Noha S.; Sticht, Heinrich; Breitinger, Hans-Georg
title: Viroporins: structure, function, and their role in the life cycle of SARS-CoV-2
date: 2022-02-24
journal: Int J Biochem Cell Biol
DOI: 10.1016/j.biocel.2022.106185
sha: 8efc3adab8f883c110c7ec3588e2dedd223b4837
doc_id: 993445
cord_uid: lfqm3iuw

Viroporins are indispensable for viral replication. As intracellular ion channels they disturb pH gradients of organelles and allow Ca(2+) flux across ER membranes. Viroporins interact with numerous intracellular proteins and pathways and can trigger inflammatory responses. Thus, they are relevant targets in the search for antiviral drugs. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underlies the world-wide pandemic of COVID-19, where an effective therapy is still lacking despite impressive progress in the development of vaccines and vaccination campaigns. Among the 29 proteins of SARS-CoV-2, the E- and ORF3a proteins have been identified as viroporins that contribute to the massive release of inflammatory cytokines observed in COVID-19. Here, we describe structure and function of viroporins and their role in inflammasome activation and cellular processes during the virus replication cycle. Techniques to study viroporin function are presented, with a focus on cellular and electrophysiological assays. Contributions of SARS-CoV-2 viroporins to the viral life cycle are discussed with respect to their structure, channel function, binding partners, and their role in viral infection and virus replication. Viroporin sequences of new variants of concern (α–ο) of SARS-CoV-2 are briefly reviewed as they harbour changes in E and 3a proteins that may affect their function.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the ongoing pandemic of Coronavirus Disease 2019. SARS-CoV-2 belongs to the genus Beta-coronavirus of the Coronaviridae family, which includes SARS-CoV and Middle East respiratory syndrome coronavirus (MERS CoV) (de Wit, van Doremalen et al. 2016) . Structural proteins S, M, and E are part of the virus envelope and mediate virushost cell attachment and entry. Coronavirusencoded accessory proteins play critical roles in virus-host interactions and modulate host immune responses, thereby participating in coronaviral pathogenicity (Lim, Ng et al. 2016) . One group of accessory proteins that have found interest as potential pharmaceutical targets are viroporins. Viroporins are small, highly hydrophobic, virus-encoded proteins that interact with membranes modifying the cell's permeability to ions or other small molecules. The name viroporin was introduced after the discovery of proteins from several different types of viruses that share common characteristics, mainly binding to intracellular proteins, and activity as ion channels (Carrasco, Perez et al. 1993) . Ion channel activity of viroporins has been described earlier, when enhanced membrane permeability was found in several virus-related cell systems (Carrasco 1978 , Gonzalez and Carrasco 2003 , McClenaghan, Hanson et al. 2020 . The main contribution of viroporins to the viral life cycle involves virion assembly and release from infected cells (Iwatsuki-Horimoto, Horimoto et al. 2006 , Lu, Zheng et al. 2006 , Steinmann, Penin et al. 2007 , Ruiz, Guatelli et al. 2010 ) as shown by viroporin defective viruses that were unable to accomplish proper folding and viral release (Terwilliger, Cohen et al. 1989 , Iwatsuki-Horimoto, Horimoto et al. 2006 , Lu, Zheng et al. 2006 , Jones, Murray et al. 2007 , Steinmann, Penin et al. 2007 . Viroporins have also been suggested to be involved in virus-induced apoptosis (O'Brien 1998).

A viroporin is a transmembrane hydrophilic pore including a selectivity filter, possessing defined charge selectivity and translocation efficiency that allows ions or small solutes to pass the host cell's membranes along their electrochemical gradient. Viroporins have been described in numerous viruses (Table 1) , and they classically participate in the viral replication cycle by locating to membranes connecting the ER lumen and cytosol. Presence of viroporins on the J o u r n a l P r e -p r o o f plasma membrane has been observed in some studies and not found in others. To date, the question remains whether viroporins allow random passage of their permeant ions, or if they are controlled by a gating mechanism similar to ligand-or voltage-gated neuronal ion channels.

Viroporins have been found in numerous RNA viruses, including E viroporins of SARS-CoV, mouse hepatitis virus, and infectious bronchitis virus, 6K protein from togaviruses, the small hydrophobic protein (SH) from paramyxoviruses, non-structural protein 4 (NSP4) from rotaviruses, p10 from avian orthoreovirus, p7 from pestiviruses and flaviviridae including human hepatitis C virus, and Kcv from Paramecium bursaria. Viroporins and membrane proteins with putative channel function were also found on DNA viruses such as the agnoprotein of JC polyomavirus, viral protein 4 of Simian virus 40 (SV40), and E5 of human papillomavirus type 16. A number of excellent reviews on channel proteins of viruses are available (Nieva, Madan et al. 2012; Gonzalez and Carrasco 2003; Scott and Griffin 2015) . Properties of the bestcharacterized viroporins are summarized in Table 1 .

Viroporins can be classified into classes I to III, named after their number of transmembrane domains. The position of the amino-and carboxyl-terminal domains can be luminal or cytosolic which determines their subgroup (Nieva, Madan et al. 2012) . Class IA viroporins contain a short luminal amino-(N) terminal domain (< 25 amino acids) and a larger carboxyl-(C) terminal domain of around 50 amino acids. Class IB viroporins possess a short cytosolic amino (N)terminal domain while its longer carboxyl (C)-terminal part is located in the endoplasmic reticulum (ER) lumen. The third class has so far only one member, namely the SARS CoV open reading frame (ORF) 3a. Here, the short N-terminal domain points to the ER lumen, followed by three transmembrane domains, hence the longer C-terminal domain is situated in the cytosol (FIG. 1A) .

In addition to viroporins, several other virus-associated glycoproteins were described to increase cell membrane permeability (Chernomordik, Chanturiya et al. 1994 , Arroyo, Boceta et al. 1995 , Newton, Meyer et al. 1997 , Ciccaglione, Marcantonio et al. 1998 . Some viral glycoproteins are J o u r n a l P r e -p r o o f able to oligomerize, thereby forming pore-like structures that allow transmembrane permeation of ions as viroporins do. In addition, domains adjacent to the transmembrane region could act destabilize membrane structure. Indeed, viroporin activity is fully replaced in the HIV-2 virus that does not express typical viroporins (Bour and Strebel 1996) . Taken together, in viruses lacking the typical viroporins their function could be replaced by pore-forming glycoproteins, while other viruses are in need of ion channel activity (Bour and Strebel 1996, Gonzalez and Carrasco 2003) . It was proposed that pore-forming glycoproteins play key roles during virus entry and in some cases during virus budding, while viroporins are essential in virus assembly and release (Gonzalez and Carrasco 2003) . It should be noted that transmembrane channel activity is essential for virus replication and proliferation, whether provided by viroporins or other pore-forming proteins. It has been suggested that viroporins help to create the negative membrane curvature that is needed for closure and release of virus-containing particles (FIG 1B) .

The maintenance of membrane gradients and the specific ionic composition within defined cellular compartments and organelles is essential for cellular homeostasis. Therefore, destruction of these finely balanced systems by viroporin activity will affect multiple cellular processes including trafficking, signaling and the induction of cell death by apoptosis (Scott and Griffin 2015) . Calcium homeostasis is disturbed when Ca 2+ leaks from intracellular stores such as mitochondria, ER and Golgi complexes, as is commonly observed after viroporin expression.

Examples are poliovirus protein 2B (Aldabe, Irurzun et al. 1997 ), picornavirus 2B proteins (de Jong, de Mattia et al. 2008 ) and rotavirus NSP4 viroporin (Diaz, Chemello et al. 2008 , Hyser, Collinson-Pautz et al. 2010 . It was further shown that the envelope proteins (E) of both, SARS-CoV, and SARS-CoV-2 form calcium selective channels, thereby triggering the activation of the NLRP3 inflammasome, leading to IL-1 overproduction (Nieto-Torres, Verdia-Baguena et al. 2015) , (Xia, Shen et al. 2021 ). Ca 2+ acts as an intracellular messenger and regulator of numerous cellular processes, so changes in calcium levels affect these processes inside both, host cells or the virus (Zhou, Frey et al. 2009 ). Host cell apoptosis is often observed after viroporin expression (Hajnoczky, Davies et al. 2003 , Aweya, Mak et al. 2013 , Breitinger, Farag et al. 2021 , and can also be induced by high calcium levels (Madan, Castello et al. 2008 ). In addition, J o u r n a l P r e -p r o o f increased calcium levels can indirectly alter processes like gene expression, viral maturation, or release (Zhou, Frey et al. 2009 , Nieva, Madan et al. 2012 . Several viroporins were described as pH-gated, and H + conducting channels, thereby they would be able to change the pH of the cytosol and organelles. It has been proposed that viroporins action might lead to an increase of cytosolic pH by de-acidification of the ER-Golgi intermediate compartment (ERGIC) . Use of this mechanism was suggested for IAV M2 (Ciampor, Bayley et al. 1992 , Ciampor, Thompson et al. 1992 , Sakaguchi, Leser et al. 1996 , HCV p7 (Wozniak, Griffin et al. 2010 , Breitinger, Farag et al. 2016 , infectious Bronchitis CoV (IBV) envelope protein (Westerbeck and Machamer 2019) and SARS CoV-2 E protein (Nieto-Torres, Verdia-Baguena et al. 2015 , Cabrera-Garcia, Bekdash et al. 2021 , Trobec 2021 . In all these cases, shunting of pH may help to protect acidlabile proteins or particles during viral release. In case of the IBV envelope protein, it is proposed, that the altered pH in the Golgi protects the IBV spike protein and helps in releasing virus particles (Westerbeck and Machamer 2019) . Notably, the cytosol constitutes the largest cellular fraction by volume (54 %), while rough and smooth ER together occupy 15 %, and lysosomes and endosomes only make 2 % of eukaryotic cell volume (Alberts, Johnson et al. 2002) . Thus, it would be expected that proton channels in ER, ERGIC, endosomes and lysosomes could change cytosolic pH only locally, and to a small extent, while the pH of these organelles and compartments themselves would be strongly altered in the presence of active viroporins.

The activation of the inflammasome by viroporins was reviewed before (Farag, Breitinger et al. 2020 ). Inflammasomes are activated upon cellular infection or stress that trigger the maturation of proinflammatory cytokines such as IL-1β to activate innate immune defenses (Latz, Xiao et al. 2013 ). The multi-protein complexes are made up of a sensor protein -the adaptor protein ASC (apoptosis-associated speck-like protein containing CARD) -and the cellular protease caspase-1 (Schroder and Tschopp 2010) . In general, caspases are cysteine proteases that initiate or execute cellular programs, leading to inflammation and/or cell death; inflammasomes activate a specific class called inflammatory caspases (Martinon, Gaide et al. 2007 ). Mammalian inflammatory J o u r n a l P r e -p r o o f caspases contain a CARD domain followed by a catalytic cysteine residue-containing domain (Boatright and Salvesen 2003) . Caspase-1 is synthesized as an inactive zymogen (pro-caspase-1) that undergoes autocatalytic processing upon appropriate stimulus (Boatright and Salvesen 2003) . Within the inflammasome, caspase-1 is activated upon interaction with the ASC adapter protein bridging the protease with NOD-like receptors (NLRs) (Nadiri, Wolinski et al. 2006) .

Several pro-inflammatory cytokines of the IL-1 family, mostly IL-1β and IL-18, play an important role in antimicrobial host defense (Dinarello 1984) . IL-1β activates the release of other proinflammatory cytokines such as TNF and IL-6 and is responsible for the acute phase response, which includes fever, acute protein synthesis, anorexia and sleepiness (Martinon, Mayor et al. 2009 ). IL-18 is produced during chronic inflammation, in autoimmune diseases, a variety of cancers, and is connected to several infectious diseases. It induces the production of multiple cytokines including IFN-γ, IL-13, IL-4, IL-8 as well as both Th1 and Th2 lymphokines (Gracie, Robertson et al. 2003) . Proinflammatory stimuli induce expression of the inactive IL-1β and IL-18 pro-forms, while cytokine maturation and release are controlled by inflammasomes (Martinon, Mayor et al. 2009 ). It is generally accepted that activation and release of IL-1β requires two distinct signals, yet the nature of these signals in the in-vivo situation during inflammation is not completely defined (Negash, Ramos et al. 2013 ) (Latz, Xiao et al. 2013) . Invitro studies characterized the first signal to be triggered by various PAMPs (pathogenassociated molecular patterns) and DAMPs (damage-associated molecular patterns), followed by Toll-like receptor (TLR) activation, which induces pro IL-1β synthesis (Negash, Ramos et al. 2013 ). The second signal is provoked by different DAMPS and PAMPS promoting NLRP3 inflammasome assembly and caspase-1-mediated activation of pro IL-1β and pro IL-18. The requirement of a second signal might provide a safety-net mechanism to ensure activation of potent inflammatory responses to happen exclusively in the presence of real inflammatory threats, such as pathogen infection and/or tissue injury (Christgen and Kanneganti 2020) . By disruption of the ionic balance of Ca 2+ and H + in the ER/Golgi compartment, several viroporins provide the second signal needed to activate host inflammasomes in processing and releasing of pro-inflammatory cytokines (FIG 2) . Viroporins triggering inflammasome activation include influenza virus M2 channel (Ichinohe, Pang et al. 2010) , the respiratory syncytial virus SH protein (Triantafilou, Kar et al. 2013 ), the encephalomyocarditis virus (EMCV) 2B (Ito, Yanagi et al. 2012 ), hepatitis C virus p7 protein (Shrivastava, Mukherjee et al. 2013, Farag, Breitinger et J o u r n a l P r e -p r o o f al. 2017), human immunodeficiency virus type 1 (HIV-1) vpu protein (Triantafilou, Ward et al. 2021 ), classical swine fever virus p7 protein (Lin, Liang et al. 2014 ) and the SARS CoV envelope protein (Nieto- , Nieto-Torres, Verdia-Baguena et al. 2015 , as well as SARS CoV 3a (Kanzawa, Nishigaki et al. 2006 , Chen, Moriyama et al. 2019 , Siu, Yuen et al. 2019 ).

Enveloped viruses enter the host cell via endocytosis, and are then released into the cytosol directly at the plasma membrane, or follow the endocytic pathway and enter the cytoplasm through early endosomes or late endosomes depending on their signals to trigger and support fusion (White and Whittaker 2016) . Transcription takes place in the cytosol (SARS CoV, HCV, RSV, PV) or in the nucleus after entering through the nuclear core complex (IAV, HIV-1).

Although some viroporins participate in different steps of the viral life cycle, such as cell entry and replication (Castano-Rodriguez, Honrubia et al. 2018) , their main activity concentrates on virion assembly and release from infected cells as shown by the fact that viruses lacking viroporins that fail to accomplish proper assembly and release (Lu, Zheng et al. 2006 , Steinmann, Penin et al. 2007 , Ruiz, Guatelli et al. 2010 . Assembly, budding and release of enveloped viruses from infected host cells follows a general mechanism which comprises several stages: (1) preparation and assembly of virion (2) budding, (3) scission and (4) viral release.

Assembly depends on expression, transport, and accumulation of viral structural proteins to cellular membranes where budding occurs. Depending on the specific virus, the zone of budding forms at the plasma membrane (IAV, HIV-1) or on cytosolic organelle membranes, for example the ERGIC (SARS CoV) or ER (HCV), or inside the infected host cell (Kien, Ma et al. 2013) .

Proteins, that are essentially involved in budding-zone formation are HA and neuraminidase for IAV (Chen, Leser et al. 2007 ), M protein for CoVs, (de Haan, Kuo et al. 1998 , de Haan, Vennema et al. 2000 , the core protein in HCV (Hourioux, Ait-Goughoulte et al. 2007 ) and gag for HIV-1 (Gheysen, Jacobs et al. 1989 ). The next stage in viral release is the formation of the viral bud. Even though budding is mainly triggered by molecular interactions of viral components interacting with core viral proteins (M1 for IAV, N protein for SARS CoV, gag for HIV and core protein for HCV) (Chen, Leser et al. 2007 ) membrane flexibility is necessary to enable positive membrane curvature. Integration of viroporins inside the appropriate membrane J o u r n a l P r e -p r o o f play an important role in this process leading to the dissipation of membrane potential. It has been suggested that M2 protein from IAV could be responsible for the curvature of cellular membranes (Rossman, Jing et al. 2010 ). Next stage in viral release comprises bud closure and scission of the viral envelope from the cellular membrane (Chen, Leser et al. 2007) . It is assumed that viroporins are involved in building the negative membrane curvature necessary for closure preceding scission and release (FIG 1B) . Best studied viroporin in this context is the M2 protein of IAV (Rossman, Jing et al. 2010) . Some viruses like HIV-1 (Garrus, von Schwedler et al. 2001 ), HCV (Ariumi, Kuroki et al. 2011 ) and herpes simplex virus type 1 (Pawliczek and Crump 2009), but not IAV (Rossman, Jing et al. 2010) , use the cellular endosomal sorting complex required for transport (ESCRT) in viral release. Specific cellular receptors or other cellular restricting factors are proposed to help with exocytosis at the end phase of viral release.

One approach towards treatment of virus-induced diseases is the identification of highly specific inhibitors. This can be achieved by testing activity against the entire virus using virus-related assays such as plaque-forming assays. A disadvantage of whole-virus assays is that the target protein itself is not detected, furthermore, high levels of biosafety are required for in vivo studies. If the search is focused on specific proteinssuch as viroporinsrecombinant expression of the gene of interest is another option. Here, no information viral activity in vivo is obtained, but the specific target protein can be studied in isolation. In this case, one needs a good, fast, and reliable testing system to guarantee high specificity for a safe preliminary selection of drug candidates. A number of assays for the study of viroporin activity have been 

The appearance of hemadsorption is dependent on the attachment of red blood cells (RBC) to the surface of cells infected with enveloped, hemagglutinin (HA)producing viruses, such as influenza, measles, or mumps. Originally developed to detect HA-virus interaction, the assay was adapted for viroporins by using an intracellular HA, which only gets translocated to the cell surface when the active viroporin disrupts vesicular pH. On this base an activity assay was developed (Shelokov, Vogel et al. 1958 , Sakaguchi, Leser et al. 1996 and used for activity testing of the IAV M2 channel (Medeiros, Escriou et al. 2001 ) and the HCV p7 protein (Griffin, Harvey et al. 2004 , Breitinger, Farag et al. 2016 ). Since the recombinant expression of viroporins induces apoptosis in the host cells, transfected cellsi.e. those that bind RBCs and generate the recorded signal are weakened or destroyed. This, together with the need of numerous washing steps render the assay challenging and ineffective (Breitinger, Farag et al. 2021) . Washing also breaks weak erythrocyte binding.

IAV M2 and HCV p7 were described as mainly H + selective channels. They target host compartments are the ER, Golgi, mitochondria and lysosomes (Nieva, Madan et al. 2012) . By integrating viroporins into their membranes, channel activity results in de-acidification, thereby equilibrating the pH of cytosol and the acidic compartments. Commercially available acidsensitive fluorescent dyes show strong fluorescence in acidic environments which is reduced upon increasing pH. Application of these dyes to untransfected control cells will give rise to emission of fluorescence from the intracellular acidic compartments. Viroporin transfected test cells will have the pH of these compartments shunted, leading to a reduced fluorescence signal.

Fluorescence based cellular assays were used for HCV p7 channels after recombinant expression on different cells (Wozniak, Griffin et al. 2010 , Breitinger, Farag et al. 2016 , Breitinger, Farag et al. 2021 . The test can be used with living cells as well as after fixation of cells with paraformaldehyde (Wozniak, Griffin et al. 2010 ).

Recently, a simple cell viability assay was introduced for activity testing on HEK293 cells expressing viroporins. Recombinant expression of active viroporin will generally weaken cells and lead to an increase in the fraction of dead cells by inducing apoptosis (Aweya, Mak et al. 2013 ). This overall loss of living cells can be detected using cellular viability assays. The method has been adapted to assay the viroporin activity (reduced cell viability) as well as function of viroporin inhibitors (cell viability restored) and can also identify cytotoxicity of potential viroporin inhibitors (reduced viability in control culture) , Breitinger, Farag et al. 2021 ).

Commercial membrane-forming lipids such as Phosphatidic acid (PA), phosphatidylcholine acid (PC) and phosphatidylethanolamine are commonly used. Lipids are dried under argon to form a film, then buffers are used to re-hydrate the lipids and form (nano)vesicles. Fluorescent dyes of varying molecular weights can be incorporated into the vesicles for later analysis. Uni-lamellar liposomes can be produced by extrusion through appropriate membrane filters. Liposomes need to be purified by centrifugation and finally resuspended in appropriate buffer (StGelais, Tuthill et al. 2007 ). 

Solvent-free planar lipid bilayers are prepared following specific protocols and sandwiched between two half glass cells. In an example procedure (Montserret, Saint et al. 2010 ), phosphatidylcholine, dissolved in hexane (0.5%), is spread on the top of an electrolyte solution (500 mM KCl, 10 mM HEPES, pH 7.4) in both compartments. Bilayer formation is achieved by lowering and raising the level in one or both compartments and monitoring capacity responses. A transmembrane voltage clamp is applied through an electrode in the cis-side, with grounding on the trans-side. Then, purified peptides are added in nM concentrations. Ion selectivity (Hodgkin and Huxley 1947) and conductance can be determined using buffers of different concentrations of permeant ions on the cis-and trans side. The method was used for the electrophysiological characterization of several viroporins (Wilson, McKinlay et al. 2004 , Torres, Maheswari et al. 2007 , Whitfield, Miles et al. 2011 , Pham, Perry et al. 2017 , Dey, Siddiqui et al. 2019 .

Two sets of experiments have been used: (I) measurement of current traces from single voltageclamped cells in the whole-cell mode that are lifted and positioned in front of a perfusion device.

Inhibitor, or buffers of varying pH conditions are then applied directly onto the cell (Chizhmakov, Ogden et al. 2003 , Breitinger, Farag et al. 2016 .

The second approach is to apply a current-voltage ramp to cells in the whole-cell configuration.

In this scenario, the cell can be studied without lifting. Since viroporin induced currents are very small and cells usually are weakened due to cell damage induced by viroporin activity, single trace measurements are challenging, especially since the procedure of detaching the cell and lifting it to the delivery system takes time and stresses the cell further, often resulting in increasing leak currents that overlay the small signal. In case of voltage ramps, currents can be recorded directly after patching and transferring cells into whole cell configuration. Even then, HEK293 cell currents under identical conditions can have a large cell to cell variability (Premkumar, Wilson et al. 2004 , Schwarz, Sauter et al. 2014 , Breitinger, Farag et al. 2021 , Cabrera-Garcia, Bekdash et al. 2021 . Thus, the method requires signals from many cells under each testing condition to be averaged since no direct comparison J o u r n a l P r e -p r o o f on single-cell basis is possible. If carefully done, these methods allow the direct study of viroporin channel behaviour under in-vivo like conditions.

The Coronaviridae family comprises four groups, named α-, β-, γ-, and δ-Coronavirus (CoV). αand -CoV mainly are mainly found in mammals, γ-CoV and δ-CoV are found in birds.

Recombination events have been described in several coronaviruses infecting humans and animals (Vakulenko, Deviatkin et al. 2021) . Phylogenetic studies suggest that bats are the major source for α-CoV and β-CoV infection of other mammals. Analysis of bat CoVs showed that α-CoV change hosts more frequently than β-CoV. However, β-CoV is more relevant as a source of new pathogenic human viruses, including SARS-CoV-2. Wild birds are the source for highly diversified γ-and δ-CoV (Vakulenko, Deviatkin et al. 2021) . Table 3 gives some examples, including all coronavirus species discussed here. (FIG 3A, 4A) . Among the new SARS-CoV-2 variants of concern, only very few single mutations were described (FIG 3D, 4A) . The exchange L21V in the α variant as well as T9I in the recent ο strain may affect channel function, while the exchange P71L observed in the β variant is directly preceding the PALS1-binding motif 72 DLLV 75 (FIG: 3D) and may have an effect on intracellular interactions mediated by the E protein.

Among all open reading frames (ORFs) of SARS CoV, ORF3a is the largest; it encodes a transmembrane proteinthe 3a proteinof 274 amino acids. While there are large differences in the sequences between SARS-CoV and SARS-CoV-2 (identity of 72.4%) (FIG 3B, 4B) , only minor exchanges are noted among the new, highly infectious SARS-CoV-2 variants (FIG 3E,   4B ). The new variants of concern contain the following mutations: α: Q57H, F120V, G172V; β: Q57H, S171L; γ: Q57H; δ: S26L (Fig. 3E ). Of these exchanges, QH results in a moderate local increase in basicity, while GV increases side chain size. Replacement SL will alter side chain size and polarity. It remains to be seen whether these exchanges result in a pronounced change of protein function and contribution to the viral life cycle.

The intact ORF8present in animal (bat) and some early human isolatesencodes a 122amino-acid polypeptide, 8ab(+), while human SARS-CoV has a deletion of 29 nucleotides, within ORF8a that results in the generation of two proteins, ORF8a and ORF8b, polypeptides of 39 and 84 residues, respectively, that appear without function (Oostra, de Haan et al. 2007 , Muth, Corman et al. 2018 . It has been shown that the 29 bp deletion causes a dramatic reduction of replication of bat SARS-CoV (Oostra, de Haan et al. 2007 , Muth, Corman et al. 2018 .

Recent SARS-CoV-2 variants possess again the complete protein from the intact ORF8. The ORF8 sequence is changing fast and uncontrolled leading to a sequence identity of less than 20% between SARS-CoV and SARS-CoV-2, this may be related to some unknown viral infection strategies (Neches, Kyrpides et al. 2021 ). The small ORF8a proteinonly present in SARS-CoV contains a single transmembrane domain which was shown to form active ion channels in artificial lipid bilayers (Chen, Kruger et al. 2011 ). ORF8b appeared not to be expressed in SARS-CoV-infected cells. Only after placing the gene immediately behind the T7 promoter, expression was observed, resulting in a soluble monomeric protein found in the cytoplasm, which was highly unstable and rapidly degraded (Oostra, de Haan et al. 2007 ). ORF8 of SARS-CoV-2 is a 121-amino acid protein consisting of an N-terminal signal sequence for endoplasmic reticulum import. In contrast to the envelope protein E, ORF3a and ORF8a, ORF8 was lacking any membrane spanning α helices in a structure determined by X-ray crystallography (Table 2, FIG 3C, 4C) . Instead, the structure revealed that the ORF8 core consisted of two antiparallel βsheets (Flower, Buffalo et al. 2021) , which questions the role of ORF8 and derived proteins as 

The first experiments on the E protein of different coronaviruses confirmed that its expression in mammalian cells alters membrane permeability (Liao, Lescar et al. 2004 , Liao, Yuan et al. 2006 ). Furthermore, the E-protein was found to be located mainly in the endoplasmic reticulum (ER) and Golgi apparatus where its ion channel activity increases the pH of the affected compartments (Nal, Chan et al. 2005 , Liao, Yuan et al. 2006 , Nieto-Torres, Dediego et al. 2011 , Cabrera-Garcia, Bekdash et al. 2021 . Before E protein structures were available, computational secondary structure predictions of the β-and γ-CoV E proteins suggested that a conserved proline residue at position 54, near the C-terminus was centered in a β-coil-β motif (Cohen, Lin et al. 2011) . This motif was considered as a putative Golgi-complex targeting signal as exchange of this proline with alanine was sufficient to disrupt the localization of a mutant chimeric protein to the Golgi complex, directing it to the plasma membrane instead (Cohen, Lin et al. 2011) . A reciprocal chimera consisting of the N-terminal and hydrophobic domains from SARS-CoV E fused to the C-terminal tail of vesicular stomatitis virus glycoprotein (VSV-G) was expressed and analyzed by immunofluorescence microscopy, indicating its localization inside the Golgi complex. This suggests that a second Golgi localization tag might be present within the Nterminal region of the E protein (Cohen, Lin et al. 2011). J o u r n a l P r e -p r o o f

The topology of the E protein was studied in microsomal membranes and mammalian cells, consistently indicating a single-spanning membrane protein. In the ER the N-terminus being translocated across the membrane to the luminal side, while the C-terminus is thus exposed to the cytoplasmic side of the ER (Duart, Garcia-Murria et al. 2020) .

When the ion channel activity of E protein was first studied, it was described as a cationselective ion channel (Wilson, McKinlay et al. 2004 ) composed of five subunits (FIG 4A) .

Further studies confirmed the E protein to be a functional ion channel in artificial membranes and in mammalian cells after recombinant expression (Pervushin, Tan et al. 2009 , Xia, Shen et al. 2021 ). Xia et al investigated the cation selectivity in more detail using purified SARS-CoV-2 E channels, reconstituted in bilayer lipid membranes. Studies of the reversal potentials in solutions with varying concentrations of different cations and Clas permeant ions showed that the channel is permeable to K + and Na + with similar efficiency. The channel is also permeable to divalent cations Ca 2+ and Mg 2+ , yet their permeability is lower than that of the monovalent ions. The permeability ratio shows a distinct selectivity pattern that is also known for classic voltage-dependent channels (Xia, Shen et al. 2021 Notably, while studies of SARS-CoV-2 E protein reconstituted in bilayer lipid membranes had

shown an ion preference of monovalent cations over divalent cations (Xia, Shen et al. 2021 

E protein channel activity, like that of other viroporins, can be blocked by various channel blockers or inhibitors. Hexamethylene amiloride (HMA) was first described to inhibit currents on S1-E (Torres, Maheswari et al. 2007 ). In a further study, amantadine and rimantadine were investigated for their inhibitory ability after recombinant E protein expression in HEK293 cells.

Both substances reduced E protein activity in cell viability assay and electrophysiological J o u r n a l P r e -p r o o f measurements, with rimantadine inhibition being five-fold more potent than that of amantadine ). Genistein and Kaempferol, two flavonoids, were described as viroporin inhibitors before (Sauter, Schwarz et al. 2014 , Schwarz, Sauter et al. 2014 . Genistein inhibited the HIV-1 viral protein U (vpu) (Sauter, Schwarz et al. 2014) while Kaempferol derivatives were shown to act as inhibitor against SARS CoV ORF 3a protein (Schwarz, Sauter et al. 2014 ) when both ion channels were expressed in Xenopus oocytes and the activity assessed using electrophysiological techniques. When different flavonoids were tested as putative E protein antagonists, Epigallocatechin and quercetin exhibited inhibitory properties at concentrations similar to rimantadine activity. This testing was done in S1-E protein expressing HEK293 cells using both, cell viability assays and patch-clamp electrophysiological analysis .

The flavonoid rutin or quercetin-3-O-rutinoside was identified as putative binding partner of SARS-CoV-2 E protein in a molecular docking study of membrane, envelope and nucleocapsid proteins of the SARS-CoV-2 (Bhowmik, Nandi et al. 2020) . Further inhibitors, that were identified using molecular docking techniques are Tretinoin (Dey, Borkotoky et al. 2020) ;

Sinapic Acid showed an in vitro inhibition of -SARS CoV-2 activity, was further studied by molecular dynamic simulations which revealed the E-protein as its major pharmacological target (Orfali, Rateb et al. 2021) . A study based on computational simulation approaches identified

Belachinal, Macaflavanone E, and Vibsanol B as putative E protein inhibitors. According to this study, binding of these compounds reduces the random motion of the SARS-CoV-2 E protein, which results in inhibition of its activity (Gupta, Vemula et al. 2021) .

Despite its role in efficient production and release of new virions, the E protein is incorporated at low levels within the viral envelope. SARS CoVs bud inside the lumen of the ERGIC followed by transport and release through the secretory pathway (Goldsmith, Tatti et al. 2004 ). Coexpression experiments in mammalian cells producing virus-like-particles (VLPs) gave dissenting results. While co-expression of SARS-CoV E protein with M and N proteins induced efficient production and release of VLPs, co-expression of E and M without N protein gave very low VLP levels (Siu, Yuen et al. 2019 ). Assembly of MHV (murine -CoV) VLPs was J o u r n a l P r e -p r o o f independent of N protein when M and E proteins were co-expressed in mammalian OST7-1 cells (Vennema, Godeke et al. 1996) . In contrast, when HEK293 cells were used, co-expression of SARS-CoV M and N structural proteins was sufficient to trigger budding of VLPs even in absence of the E protein (Huang, Yang et al. 2004 , Hatakeyama, Matsuoka et al. 2008 ).

However, secretion of these VLPs was inefficient (Hatakeyama, Matsuoka et al. 2008) . A recombinant SARS-CoV lacking the E protein showed reduced virus expression and the process of virus release was less efficient than for the wild-type analogue (DeDiego, Alvarez et al. 2007 ).

Budding and exocytosis of virus requires bending of membranes to form vesicles. A potential role of the E protein in inducing membrane curvature has been proposed but still needs further investigation. The SARS-CoV E protein was suggested to build a transmembrane helical hairpin after incorporation into dimyristoylphosphocholine (DMPC) lipid vesicles which was assumed to deform lipid bilayers leading to an increase in their curvature (Arbely, Khattari et al. 2004 ).

However, this helical transmembrane hairpin has not been confirmed in NMR structures (Pervushin, Tan et al. 2009 , Li, Surya et al. 2014 , Surya, Li et al. 2018 . (Teoh, Siu et al. 2010 , Toto, Ma et al. 2020 , Chai, Cai et al. 2021 , Javorsky, Humbert et al. 2021 . PALS1 is a tight junction-associated protein that plays a crucial role in establishing and maintaining epithelial polarity in various organisms. It was suggested that the interaction of PALS1 with the SARS-CoV E protein is involved in the disruption of the lung epithelium in SARS patients (Teoh, Siu et al. 2010 Toll-like receptors (TLRs) are mediators of inflammatory responses and cytokine release. It was shown that infection with SARS-CoV-2 resulted in an upregulation of Myd88, a TLR adaptor protein, and TLRs 1, 2, 4 5, and 8 (Zheng, Karki et al. 2021) . Expression of these mediators increased with clinical severity of SARS (Zheng, Karki et al. 2021) . Independent of the PBM -E protein interactions, TLR2 was identified as a direct E protein ligand, and blocking of the TLR2 pathway resulted in protection against SARS infections (Zheng, Karki et al. 2021) . Expression of

Bcl-xL was indeed demonstrated in vitro and in vivo (Yang, Xiong et al. 2005) . Two further SARS-CoV E protein interaction partners were identified by mass spectrometry: Na + /K + -ATPase, the main cellular ion pump involved in ion homeostasis control and stomatin, an ion channel regulator (Nieto- Torres, Dediego et al. 2011) . In the search for new physiological targets for antiviral drugs, a screen for interactions between 26 SARS-CoV-2 proteins and human host cells suggested a total of 6 interactions for the E protein (Gordon, Jang et al. 2020 ).

These included Bromodomain and Extra-terminal domain (BET) proteins BRD2 and BRD4, which J o u r n a l P r e -p r o o f control the expression of angiotensin converting enzyme 2 (ACE2) that represents the docking site for SARS CoVs on host cells. A second proposed interaction partner is the beta subunit of the adaptor-related protein complex (AP3B1), a protein involved in protein sorting in the late Golgi network. Other putative binding partners include the cyclophilin CWC27 as well as ZC3H18, involved in mRNA biogenesis, and SLC44A2 which regulates choline transport into mitochondria, thus affecting platelet aggregation exerting an influence on thrombosis (Gordon, Jang et al. 2020) .

Post-translational modification of coronaviruses was reviewed in 2018 with consideration of different postulated membrane topologies. Minor N-linked glycosylation is described on residue N66 in a membrane topology where the C-terminus is exposed to the luminal side of the ER (Fung and Liu 2018) . However, the widely accepted topology of the E protein, which is in agreement with NMR structures shows the N-terminus located at the luminal side, while the Cterminusincluding N66is directed towards the cytosol, this membrane topology would not be amenable to glycosylation. The three cysteine residues C40, C43 and C44 in SARS-CoV E protein are modified by palmitoylation (Liao, Yuan et al. 2006, Fung and Liu 2018) , which might play a role in its subcellular trafficking and association with lipid rafts. When plasmid DNAs encoding mouse hepatitis CoV (MHV) S, E, M, and N was introduced in HEK293 cells and compared to experiments where E protein residues C40, C44 and C47 were mutated to alanine, VLP formation was significantly reduced (Boscarino, Logan et al. 2008 ). Lopez et al exchanged the same cysteine residues with alanine in MHV E protein which resulted in crippled virus morphology and reduced yields, as well as faster E protein degradation (Lopez, Riffle et al. 2008) . Taken together, palmitoylation of MHV E protein seems to stabilize the protein and its biological activity during assembly of mature virions. Surprisingly, in E and N protein coexpression experiments, palmitoylation of SARS-CoV E protein had no effect on its association with N protein or subsequent VLP production, and thus is not required for SARS-CoV assembly, and the contribution of E protein palmitoylation to virus packaging and VLP production appear independent form each other (Tseng, Wang et al. 2014) . Molecular dynamics simulations of SARS CoV-2 E protein indicate that the stability of the palmitoylated E protein structure is J o u r n a l P r e -p r o o f increased, while loss of palmitoylation causes reduction of the pore radius, and even collapse of the pore." (Sun, Karki et al. 2021) .

Deletion of the E protein in general represses virus growth in culture and abrogates virulence in animals due to its reduced inflammatory reaction. Infections with mutated viruses where E protein function was repressed or deleted resulted in very mild or almost absent clinical symptoms while production of anti-inflammatory cytokines and T-cell responses were still robust (Regla-Nava, Nieto- . Several studies performed in mice concentrated on SARS-CoV-ΔE approaches with the goal to find successful vaccine candidates (Netland, DeDiego et al. 2010 , Fett, DeDiego et al. 2013 , and the E protein is considered a promising target for vaccine development.

ORF 3a is the largest unique open reading frame in the SARS CoV genome, encoding a protein of 274 amino acids. The ORF 3a protein forms an ion channel (Lu, Zheng et al. 2006) , induces vesicle formation (Freundt, Yu et al. 2010) and modulates virus release (Lu, Zheng et al. 2006 ).

The 3a protein belongs among group 3 of viroporins (FIG 1B) possessing three transmembrane domains with the N-terminus located at the luminal side of the ER and the C-terminal domain directed to the cytosol (FIG 4B) . After the E protein, the 3a protein of SARS-CoV was shown to be involved in membrane rearrangement (Yuan, Li et al. 2005 , Freundt, Yu et al. 2010 ) and

acting as an ion channel (Lu, Zheng et al. 2006 ). Expression of S1-3a in Xenopus oocytes resulted in a homotetrameric complex, stabilized by disulfide bonds between subunits that conducted K + ions in two-electrode voltage clamp measurements (Lu, Zheng et al. 2006) . Studies on ion selectivity of SARS-CoV-2 3a channels reconstituted in proteo-liposomes revealed nonselective cation channel activity with a permeability order of Ca 2+ > K + > Na + > NMDG + (Kern, Sorum et al. 2021) . Staining and 45 Ca overlay studies on an E. coli expressed cytoplasmic domain of S1-3a showed that calcium binding introduced a change in conformation which was detected using fluorescence spectroscopy and circular dichroism (Minakshi, Padhan et al. 2014 ).

Expression of the 3a protein in various in vitro systems indicates that it mainly localizes in the J o u r n a l P r e -p r o o f

Golgi region (Padhan, Tanwar et al. 2007 , Yuan, Yao et al. 2007 ), but was also detected in the ER (Law, Wong et al. 2005) , late endosomes (Freundt, Yu et al. 2010 , Yue, Nabar et al. 2018 , Miao, Zhao et al. 2021 , lysosomes (Yue, Nabar et al. 2018 , Zhang, Sun et al. 2021 , and the trans-Golgi network (Yue, Nabar et al. 2018) .

To date, only few reports on inhibition of ORF 3a ion channel functional have been published.

Electrophysiological data on heterologously expressed 3a protein in Xenopus oocytes revealed inhibition of 3a induced currents by Kaempferol derivatives (Schwarz, Sauter et al. 2014 ) and

Emodin (Schwarz, Wang et al. 2011) . A molecular docking study on 23 compounds and subsequent experimental testing using fluorescence and UV-Vis spectroscopy identified chlorin, iron(III) protoporphyrin and protoporphyrin as a putative binding partners of ORF 3a (Lebedeva, Gubarev et al. 2021) . In a drug repurposing approach, bacteria-based functional assays that measured growth retardation, K + uptake, and change of cytoplasmic pH of E. coli that overexpressed the 3a protein of SARS-CoV-2 were tested (Tomar, Krugliak et al. 2021 ). Using these screens, a library of drugs approved for human use an approved human drugs was screened, identifying several potential channel blockers of the 3a channel, namely Capreomycin, Pentamidine, Spectinomycin, Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine and Fludarabine (Tomar, Krugliak et al. 2021) .

The general involvement of the SARS-CoV ORF3a (S1-3a) in inducing pro-apoptosis was described in several cell lines and it seems to be dependent upon its channel function (Law, Wong et al. 2005 , Chan, Tsoi et al. 2009 , Freundt, Yu et al. 2010 , Ren, Shu et al. 2020 . Several approaches were followed to better explain the role of S1-3a in pro-apoptotic function. It was shown that the cytoplasmic domain of S1-3a activates the mitochondrial p38 MAP kinase death pathway, which could be repressed by a p38 MAPK inhibitor (Padhan, Minakshi et al. 2008) . ER stress in general can cause modulation by different pathways of the unfolded protein response (UPR). Studies on S1-3a-expressing cells show that only one pathway is activated by 3a, namely the PKR-like ER kinase (PERK) pathway (Minakshi, Padhan et al. 2009 ). Involvement of 3a protein in inflammatory responses was shown as the 3a protein contributes to assembly of the NLRP3 inflammasome (Chen, Moriyama et al. 2019) . Oligomerization of 3a is mediated by Receptor Interacting Protein 3 (Rip3), which leads to lysosomal damage, caspase-1 activation, J o u r n a l P r e -p r o o f and subsequent necrotic cell death (Yue, Nabar et al. 2018) . Another aspect of 3a-induced inhibition of cell proliferation could be the observation that S1-3a protein reduces cyclin D3 expression, thereby inhibiting Rb phosphorylation, which subsequently leads to an arrest in the G1 phase of the cell cycle (Yuan, Yao et al. 2007 ).

In general, autophagy is one of the major defense mechanisms of cells in the fight against pathogens (Deretic, Saitoh et al. 2013) . However, viruses have developed mechanisms to interfere with the autophagic process, even including autophagy for their own replication.

Coronaviruses were suggested to interact with the autophagy pathway (Carmona-Gutierrez, Bauer et al. 2020 ) and involvement of S2-3a protein in this process was investigated. Fusion of autophagosomes with lysosomes is prevented by S2-3a, located in the late endosome, blocking interaction of the homotypic fusion and protein sorting (HOPS) complex with the SNARE complex and RAB7 which is required for autolysosome formation, thereby inhibiting fusion of autophagosomes with lysosomes (Miao, Zhao et al. 2021 , Zhang, Sun et al. 2021 . A further study confirms that 3a of SARS-CoV-2 alone is able to induce incomplete autophagy in infected cells. ORF3a interacts with autophagy regulator UVRAG, inducing PI3KC3-C1 activity, and at the same time inhibits PI3KC3-C2 (Qu, Wang et al. 2021) . Surprisingly, despite the high similarity of 72.2 % between S1-3a and S2-3a, the cellular autophagy response could only be observed for S2-3a (Qu, Wang et al. 2021) .

ORF3a colocalizes with the M protein in the Golgi compartment, the E protein is also mainly localized in the ERGIC compartment. However, aside of this localization in the same compartment, there is no strong evidence that these virus proteins interact with each other. Some interactions of ORF 3a with proteins involved in autophagy were described in the section 'Apoptosis and autophagy'. Using biochemical, biophysical and genetic techniques, an interaction of the S1-3a with caveolin-1 was identified (Padhan, Tanwar et al. 2007 ). Caveolins (1-3) are 21-24 kDa proteins building the major structural component of caveolae, a special type of lipid raft responsible in the uptake of small molecules through glycosylphosphatidylinositol (GPI)anchored receptors; additionally, caveolae play a role in neurotransmitter signaling (Anderson 1998) .

Pulse-chase analysis on the SARS-CoV 3a protein points to its posttranslational modification through O-glycosylation which was confirmed by an in-situ O-glycosylation assay and the absence of O-linked sugars after substitution of serine and threonine residues in the S1-3a ectodomain (Oostra, de Haan et al. 2006) .

In animal isolates and early human isolates, the SARS-CoV encoded a single protein 8ab.

However, in later human isolates during the peak of 2003 SARS-CoV epidemic, a 29-nt deletion in the center split ORF8 into two smaller ORFs, encoding proteins 8a and 8b (Oostra, de Haan et al. 2007 , Muth, Corman et al. 2018 . During the SARS CoV-2 epidemic, however, ORF8ab

instead of 8a and 8b are expressed. The intact ORF8 contains a cleavable signal sequence, directing the precursor to the ER and mediating its translocation into the lumen. The cleaved protein is N-glycosylated, and remains stable in the ER (Oostra, de Haan et al. 2007 ). ORF8

activates IL-17 signaling pathway and promotes the expression of pro-inflammatory factors thereby contributing to the observed cytokine storm (Lin, Fu et al. 2021) , independent on viroporin activity. The structure of ORF8 adopts an Ig-like fold, and forms a disulfide-linked homodimer containing an intermolecular parallel -sheet (Flower, Buffalo et al. 2021 ).

The 8a polypeptide (39 amino acids) of SARS-CoV was detected in mitochondria, and its overexpression results in increased mitochondrial transmembrane potential, production of reactive oxygen species, increased caspase 3 activity, and cellular apoptosis (Chen, Ping et al. 2007 ). When reconstituted into artificial lipid bilayers 8a forms cation-selective ion channels, and computational modelling studies in a hydrated POPC lipid bilayer on a truncated 22 amino acid transmembrane helix predicted a putative homo-oligomeric helical bundle model (Chen, Kruger et al. 2011 J o u r n a l P r e -p r o o f

The genome of SARS-CoV-2 encodes for 29 proteins, of which the E-and ORF3a protein have been identified as viroporins that participate in viral replication through their channel activity and through interaction with intracellular proteins and pathways. Viroporin activity is indispensable for virus replication. Techniques for the study of activity as well as inhibition of viroporins have been developed making them a highly relevant object of study and a promising target for the development of antiviral drugs. All selected viruses are enveloped (exception: picornaviridae), single-stranded +RNA viruses except HIV-1 (reverse transcribing RNA virus). Ion selectivities have been adapted from a previous review (Scott and Griffin 2015) except HCV p7 (Montserret, Saint et al. 2010) , and SARS CoV-2 E protein (Xia, Shen et al. 2021 ) and ORF 3a (Kern, Sorum et al. 2021) proteins. §only modeled structures available **Rhinolophus sinicus (Chinese rufous horseshoe bat); Rhinolophus ferrumequinum (Greater horseshoe bat). J o u r n a l P r e -p r o o f Table 2 ). Conserved residues are highlighted in yellow, differences between ORF8 has highest similarity with Bat-WIV1 (57.9% identity) and Bat-HKU3 (57.0% identity).

(D,E) Sequence comparison of new variants of SARS-CoV-2 that were classified by the WHO as 'variants of concern' (SARS-CoV-2 α, β, γ, δ, ο). New mutations are highlighted in purple.

Among the SARS-CoV-2 variants, E protein (D) and 3a protein (E) show similar tendency towards developing new mutations. Three mutations were detected in E protein variants on a length of 75 amino acids, while six new mutations are described for 3a variants in a protein of 275 amino acids. Relative to length of the protein, the probability of mutation is even higher in the E protein than in 3a.

Structures of SARS-CoV-2 Viroporins

Subunits are colored in red/blue and residues that are variable between SARS-CoV, bat species

CoVs and SARS-CoV-2 are highlighted in yellow/cyan; the sites of mutation in variants of concern of SARS-CoV-2 are indicated in grey colour and spacefill and indicated in the structures. See Figure 3 for the respective sequence alignments and Table 1 between SARS-CoV and SARS-CoV-2 (yellow/cyan), exchanges found in variants of concern of SARS-CoV-2 are located at or near the pore domains and might affect channel function.

Exchange S26L is located outside the resolved structure of the protein. (C) ORF 8 protein (based on pdb 7jtl). Two subunits (red/blue) are shown. The structure shows no similarity correlation to known channel-forming proteins. The low number of conserved (red/blue) compared to J o u r n a l P r e -p r o o f nonconserved (yellow/cyan) residues reflects that ORF 8 protein sequences differ significantly between SARS-CoV, bat CoVs, and SARS-CoV-2. for virus assembling, budding and exocytosis. In this process, the E protein could bind to PALS1

and ZO-1, hindering them from trafficking to TJ. The right scheme describes possible virus dissemination after TJ disruption. Loss of the TJ transmembrane proteins PALS1 and ZO-1 lead to a progressive decomposition of the TJ and a sub-sequent leakage between neighbouring epithelial cells. As a further consequence, barrier function is lost, and SARS CoV virions can leak into underlying tissues by systemic circulate-on. This process could be the cause of severe alveolar damages observed in SARS CoV-infected patients (Teoh, Siu et al. 2010 ).

J o u r n a l P r e -p r o o f

Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus

Membrane permeabilization by poliovirus proteins 2B and 2BC

Poliovirus protein 2BC increases cytosolic free calcium concentrations

The envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein 3 and is ubiquitinated

The caveolae membrane system

A highly unusual palindromic transmembrane helical hairpin formed by SARS coronavirus E protein

The ESCRT system is required for hepatitis C virus production

Membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41

The p7 protein of the hepatitis C virus induces cell death differently from the influenza A virus viroporin M2

Identification of potential inhibitors against SARS-CoV-2 by targeting proteins responsible for envelope formation and virion assembly using docking based virtual screening, and pharmacokinetics approaches

Mechanisms of caspase activation

Envelope protein palmitoylations are crucial for murine coronavirus assembly

The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles

The human immunodeficiency virus (HIV) type 2 envelope protein is a functional complement to HIV type 1 Vpu that enhances particle release of heterologous retroviruses

Inhibition of SARS CoV Envelope Protein by Flavonoids and Classical Viroporin Inhibitors

Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors

Cell viability assay as a tool to study activity and inhibition of hepatitis C p7 channels

The envelope protein of SARS-CoV-2 increases intra-Golgi pH and forms a cation channel that is regulated by pH

Host PDZ-containing proteins targeted by SARS-CoV-2

Digesting the crisis: autophagy and coronaviruses

Membrane leakiness after viral infection and a new approach to the development of antiviral agents

Regulation of Gene Expression in Animal Viruses

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

Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis

Structural basis for SARS-CoV-2 envelope protein recognition of human cell junction protein PALS1

The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function

The p7 protein of hepatitis C virus forms structurally plastic, minimalist ion channels

Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmidderived virus-like particles

ORF8a of SARS-CoV forms an ion channel: experiments and molecular dynamics simulations

Open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis

Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome

Expression and membrane integration of SARS-CoV E protein and its interaction with M protein

An amphipathic peptide from the C-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes

Differences in conductance of M2 proton channels of two influenza viruses at low and high pH

Inflammasomes and the fine line between defense and disease

Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans Golgi compartment

Regulation of pH by the M2 protein of influenza A viruses

Hepatitis C virus E1 protein induces modification of membrane permeability in E. coli cells

Evidence for the formation of a heptameric ion channel complex by the hepatitis C virus p7 protein in vitro

Identification of a Golgi complex-targeting signal in the cytoplasmic tail of the severe acute respiratory syndrome coronavirus envelope protein

Three-dimensional structure and interaction studies of hepatitis C virus p7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine by solution nuclear magnetic resonance

Coronavirus particle assembly: primary structure requirements of the membrane protein

Assembly of the coronavirus envelope: homotypic interactions between the M proteins

Functional analysis of picornavirus 2B proteins: effects on calcium homeostasis and intracellular protein trafficking

SARS and MERS: recent insights into emerging coronaviruses

A severe acute respiratory syndrome coronavirus that lacks the E gene is attenuated in vitro and in vivo

Autophagy in infection, inflammation and immunity

In silico identification of Tretinoin as a SARS-CoV-2 envelope (E) protein ion channel inhibitor

The effect of amantadine on an ion channel protein from Chikungunya virus

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

Interleukin-1 and the pathogenesis of the acute-phase response

SARS-CoV-2 envelope protein topology in eukaryotic membranes

Viroporins and inflammasomes: A key to understand virus-induced inflammation

The p7 viroporin of the hepatitis C virus contributes to liver inflammation by stimulating production of Interleukin-1β

Complete protection against severe acute respiratory syndrome coronavirus-mediated lethal respiratory disease in aged mice by immunization with a mouse-adapted virus lacking E protein

Structure of SARS-CoV-2 ORF8, a rapidly evolving immune evasion protein

The open reading frame 3a protein of severe acute respiratory syndrome-associated coronavirus promotes membrane rearrangement and cell death

Post-translational modifications of coronavirus proteins: roles and function

The small hydrophobic protein of the human respiratory syncytial virus forms pentameric ion channels

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding

Development and validation of a high-throughput screening assay for the hepatitis C virus p7 viroporin

Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells

Ultrastructural characterization of SARS coronavirus

Viroporins

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Interleukin-18

A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria

In-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel

Calcium signaling and apoptosis

Dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (N) and membrane (M) proteins of SARS coronavirus

Potassium leakage from an active nerve fibre

Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane

Generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production

Oligomerization of the human immunodeficiency virus type 1 (HIV-1) Vpu protein--a genetic, biochemical and biophysical analysis

Rotavirus disrupts calcium homeostasis by NSP4 viroporin activity

Influenza virus activates inflammasomes via its intracellular M2 ion channel

Encephalomyocarditis virus viroporin 2B activates NLRP3 inflammasome

The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly

Structural basis of coronavirus E protein interactions with human PALS1 PDZ domain

The PDZ-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis

Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus

Augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/X1 and 7a/X4 proteins through NF-kappaB activation

Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs

Viroporins: Differential Functions at Late Stages of Viral Life Cycles. Microbial Pathogenesis: Infection and

Expression of poliovirus nonstructural proteins in Escherichia coli cells. Modification of membrane permeability induced by 2B and 3A

Activation and regulation of the inflammasomes

The 3a protein of severe acute respiratory syndromeassociated coronavirus induces apoptosis in Vero E6 cells

Theoretical and experimental study of interaction of macroheterocyclic compounds with ORF3a of SARS-CoV-2

Structure of a conserved Golgi complextargeting signal in coronavirus envelope proteins

Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability

Viroporin activity of SARS-CoV E protein

Biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein

Human Coronaviruses: A Review of Virus-Host Interactions

ORF8 contributes to cytokine storm during SARS-CoV-2 infection by activating IL-17 pathway

Classical swine fever virus and p7 protein induce secretion of IL-1beta in macrophages

Importance of conserved cysteine residues in the coronavirus envelope protein

Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release

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

Viroporins from RNA viruses induce caspasedependent apoptosis

Structure and Drug Binding of the SARS-CoV-2 Envelope Protein in Phospholipid Bilayers

NALP inflammasomes: a central role in innate immunity

The inflammasomes: guardians of the body

Coronavirus Proteins as Ion Channels: Current and Potential Research

Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes

ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS complex-mediated assembly of the SNARE complex required for autolysosome formation

The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor

The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain

NMR structure and ion channel activity of the p7 protein from hepatitis C virus

Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission

The inflammatory caspases: key players in the host response to pathogenic invasion and sepsis

Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E

Orf8 from Orf7a within the Coronavirus Lineage Suggests Potential Stealthy Viral Strategies in Immune Evasion

IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease

Immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in E protein protects against lethal respiratory disease

Rotavirus nonstructural glycoprotein NSP4 alters plasma membrane permeability in mammalian cells

Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein

Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis

Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome

Viroporins: structure and biological functions

Viruses and apoptosis

Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M

The 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8

Sinapic Acid Suppresses SARS CoV-2 Replication by Targeting Its Envelope Protein

Severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 MAP kinase activation

Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin

Molecular dynamics simulations reveal the HIV-1 Vpu transmembrane protein to form stable pentamers

Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1

Herpes simplex virus type 1 production requires a functional ESCRT-III complex but is independent of TSG101 and ALIX expression

Structure and inhibition of the SARS coronavirus envelope protein ion channel

The Rotavirus NSP4 Viroporin Domain is a Calcium-conducting Ion Channel

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

ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication

Severe acute respiratory syndrome coronaviruses with mutations in the E protein are attenuated and promising vaccine candidates

The ORF3a protein of SARS-CoV-2 induces apoptosis in cells

Influenza virus M2 protein mediates ESCRT-independent membrane scission

The Vpu protein: new concepts in virus release and CD4 down-modulation

The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus

pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus

Genistein as antiviral drug against HIV ion channel

Structure and mechanism of the M2 proton channel of influenza A virus

The inflammasomes

Kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus

Emodin inhibits current through SARS-associated coronavirus 3a protein

Viroporins: structure, function and potential as antiviral targets

Hemadsorption (adsorption-hemagglutination) test for viral agents in tissue culture with special reference to influenza

SARS-CoV-2 Envelope (E) Protein Interacts with PDZ-Domain-2 of Host Tight Junction Protein ZO1

Hepatitis C virus induces interleukin-1beta (IL-1beta)/IL-18 in circulatory and resident liver macrophages

Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC

Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions

Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system

Structural basis for the function and inhibition of an influenza virus proton channel

Computational Study on the Function of Palmitoylation on the Envelope Protein in SARS-CoV-2

Structural model of the SARS coronavirus E channel in LMPG micelles

The SARS coronavirus E protein interacts with PALS1 and alters tight junction formation and epithelial morphogenesis

Functional role of human immunodeficiency virus type 1 vpu

Blockers of the SARS-CoV-2 3a Channel Identified by Targeted Drug Repurposing

Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein

The transmembrane oligomers of coronavirus protein E

Comparing the binding properties of peptides mimicking the Envelope protein of SARS-CoV and SARS-CoV-2 to the PDZ domain of the tight junction-associated PALS1 protein

Human respiratory syncytial virus viroporin SH: a viral recognition pathway used by the host to signal inflammasome activation

Differential recognition of HIV-stimulated IL-1beta and IL-18 secretion through NLR and NAIP signalling in monocyte-derived macrophages

The role of the SARS-CoV-2 envelope protein as a pH-dependent cation channel

SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production

Modular Evolution of Coronavirus Genomes

Nucleocapsid-independent assembly of coronavirus-like particles by coexpression of viral envelope protein genes

Transport mechanisms of SARS-CoV-E viroporin in calcium solutions: Lipid-dependent Anomalous Mole Fraction Effect and regulation of pore conductance

Analysis of SARS-CoV E protein ion channel activity by tuning the protein and lipid charge

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

The Infectious Bronchitis Coronavirus Envelope Protein Alters Golgi pH To Protect the Spike Protein and Promote the Release of Infectious Virus

Fusion of Enveloped Viruses in Endosomes

The influence of different lipid environments on the structure and function of the hepatitis C virus p7 ion channel protein

Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication

SARS coronavirus E protein forms cation-selective ion channels

Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target

Bcl-xL inhibits T-cell apoptosis induced by expression of SARS coronavirus E protein in the absence of growth factors

Subcellular localization and membrane association of SARS-CoV 3a protein

G1 phase cell cycle arrest induced by SARS-CoV 3a protein via the cyclin D3/pRb pathway

SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death

The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes

TLR2 senses the SARS-CoV-2 envelope protein to produce inflammatory cytokines

Viral calciomics: interplays between Ca2+ and virus

Support by Science and Technology Development Fund (STDF) Egypt grant No 45420 to NS, UB and HGB is gratefully acknowledged.

The authors report no conflict of interest regarding this work.

UB: conception, draft, writing, proofreading; NS: draft, proofreading; HS: conception, draft, writing, proofreading; HGB: conception, draft, writing, proofreading