Viroporins are an increasingly recognized class of small viral membrane proteins (~60-120 amino acids) which oligomerize to produce hydrophilic pores at the membranes of virus-infected cells . The existence of ‘viroporins’ was proposed more than 30 years ago after observing enhanced membrane permeability in infected cells . These proteins form oligomers of defined size, and can act as proton or ion channels, and in general enhancing membrane permeability in the host . Even though viroporins are not essential for the replication of viruses, their absence results in attenuated or weakened viruses or changes in tropism (organ localization) and therefore diminished pathological effects [4, 5].
In addition to having one – sometimes two – α-helical transmembrane (TM) domain(s), viroporins usually contain additional extramembrane regions that are able to make contacts with viral or host proteins. Indeed, the network of interactions of viroporins with other viral or cellular proteins is key to understand the regulation of viral protein trafficking through the vesicle system, viral morphogenesis and pathogenicity.
In general, viroporins participate in the entry or release of viral particles into or out of cells, and membrane permeabilization may be a desirable functionality for the virus. Indeed, several viral proteins that are not viroporins are known to affect membrane permeabilization, e.g., A38L protein of vaccinia virus, a 33-kDa glycoprotein that allows Ca2+ influx and induces necrosis in infected cells . In viruses that lack typical viroporins, their function may be replaced by such pore-forming glycoproteins. For example, HIV-2 lacks typical viroporins, and ROD10 Env is an envelope glycoprotein that enhances viral particle release. In HIV-1, this function is attributed to the viroporin Vpu .
An important point that needs to be established, in view of the observed channel activity of viroporins, is whether the channels they form are selective, with a controlled gating mechanism, or whether permeabilization is non selective, like in some antimicrobial peptides . Viroporins have also been found to modulate endogenous cellular channels [9-12] and this activity may also have an important regulatory role during the life cycle of the virus.
Viroporins can be found in all kinds of viruses, RNA, DNA, enveloped and non-enveloped. Examples of viroporins are picornavirus 2B , alphavirus 6K [14-16], HIV-1 Vpu [17, 18], influenza virus A M2, (also called AM2) , RSV SH protein , p10 protein of avian reovirus , Human hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) p7 [22, 23], Paramecium bursaria chlorella virus (PBCV-1) Kcv , and coronavirus envelope proteins, e.g., SARS-CoV E [25, 26]. Recent reviews [27, 28] provide more examples and possible functional roles.
The most extensively studied viroporin to date is probably the M2 protein from influenza A virus (AM2). AM2 protein is 97-residue long, with one transmembrane (TM) domain and a C-terminal cytoplasmic amphiphilic helix. AM2 forms homotetramers and is located in the viral envelope, where it enables protons from the endosome to enter the viral particle (virion). This lowers the pH inside the viral particle, causing dissociation of the viral matrix protein M1 from the ribonucleoprotein RNP, uncoating of the virus and exposure of the content to the cytoplasm of the host cell. AM2 also delays acidification of the late Golgi in some strains [29, 30].
The proton channel activity of AM2 can be inhibited by antiviral drugs amantadine and rimantadine, which block the virus from taking over the host cell. Two different high-resolution structures of truncated forms of AM2 have been reported: the structure of a mutated form of its TM region (residues 22-46) , and a slightly longer form (residues 18-60) containing the TM region and a segment of the C-terminal domain [32, 33]. These studies suggest that the known AM2 adamantane inhibitors, amantadine and rimantadine, act by either blocking the pore [31, 34] or by an allosteric mechanism . New AM2 inhibitors have been reported , but their effectiveness against adamantane-resistant viruses remains to be established. The use of these drugs presents a classical example of targeting viral channels to treat viral infection infection [31, 32, 36].
The case of AM2 protein in influenza A represents a link between viroporin activity and structure to viral pathogenesis. Unfortunately, for many viroporins even rudimentary structural models are lacking due to high hydrophobicity, conformational flexibility and tendency to aggregate. For some viroporins however, increasing degrees of structural information can be obtained due to availability of high quality purified protein. Examples of these are the viroporins present in coronaviruses (CoV) and in the respiratory syncytial virus (RSV), envelope (E) protein and the small hydrophobic (SH) protein, respectively. Both types of virus infect the upper and lower respiratory tract of humans, and their viroporins are the subject of this chapter.
2. Envelope (E) and Small Hydrophobic (SH) proteins in respiratory viruses
2.1. SH protein in hRSV
There is currently no effective vaccine available to prevent hRSV infection. Development of vaccines has been complicated by the fact that host immune responses appear to play a significant role in the pathogenesis of the disease . Naturally acquired immunity to hRSV is neither complete nor durable, and recurrent infections occur frequently during the first three years of life. Palivizumab, a humanized monoclonal antibody directed against hRSV surface fusion F protein (Synagis, by MedImmune), is moderately effective but very expensive. It is currently available as prophylactic drug for infants at high risk. Cost of prevention limits its use in many parts of the world. The only licensed drug for use in infected people is ribavirin, but its efficacy is limited. Antibodies against both F (fusion) and G (attachment) proteins have been found in the serum of hRSV infected patients, but only provide temporary protection. Therefore, low immunoprotection and lack of suitable antivirals leads towards the search and characterization of new drug targets for the effective treatments of hRSV infection. A possible suitable target is the SH protein as will be elaborated below.
hRSV also contains six internal structural proteins: the matrix (M) protein, which provides structure for the virus particle, nucleoprotein (N), phosphoprotein (P) and large (L) polymerase protein form the ribonucleoprotein (RNP) complex, which encapsidates the RSV genome and functions as the RNA-dependent RNA polymerase. Lastly, two isoforms of matrix protein 2 (M2-1 and M2-2) are accessory proteins that control transcription and replication . Viral proteins traffic to the apical surface of polarized epithelial cells, where they assemble into virus filaments at the plasma membrane , although the mechanisms that drive assembly into filaments and budding are not well understood.
Generation of nascent hRSV genomic RNA appears to occur in discrete cytoplasmic inclusion bodies that contain the hRSV N, P, L, M2-1 and M2-2 proteins but not the F, G, or SH proteins . It is suspected that the RNP complexes form in the inclusions and then traffic to the apical membrane, where they meet with the surface glycoproteins F, G, and SH arriving from the Golgi apparatus through the secretory pathway . hRSV proteins and viral RNA assemble into virus filaments at the cell surface. These filaments are thought to contribute to cell-cell spread of the virus and morphologically resemble the filamentous form of virions seen in electron microscopy (EM) studies of virus produced in polarized cells .
In addition to interactions with viral proteins, the fact that SH proteins of hRSV and parainfluenza virus 5 (PIV5) are necessary for the inhibition of tumor necrosis factor alpha (TNF-α)-induced apoptosis [56, 63] also suggests a possible interaction with host proteins, although this has not been confirmed experimentally. However, in another study, deletion of SH protein gene from RSV did not result in increased apoptosis in infected H441 cells .
SH protein after cross-linking has been shown to form multiple oligomers of increasing size in SDS [66, 68]. Later, we showed that the TM domain of SH protein forms only homopentamers in perfluoro-octanoic acid (PFO) gels . Reports using purified full-length SH protein have confirmed the pentameric nature of the oligomer formed by this protein. For example, a bundle formation of a tagged SH protein construct was visualized under electron microscopy and was interpreted as a pentameric or a hexameric structure . Using a purified tag-free SH protein, we have unequivocally demonstrated the homo-pentameric nature of these oligomers in a variety of detergents using analytical ultracentrifugation and electrophoresis (Fig. 1) . Indeed, in the presence of PFO (Fig. 1B) and a variety of other detergents under Blue-native gel electrophoresis (Fig. 1C), the full-length SH protein migrates as a single band with a molecular weight ~40 kDa, consistent with a pentameric oligomer. The pentameric form of SH has been further confirmed by analytical ultracentrifugation sedimentation equilibrium in detergents DPC, C14SB and C8E5 micelles. In these detergents, the species distribution profiles show a best fit to a monomer-pentamer self-association model (Fig. 1 D-E).
One of the two histidines, His22, was suggested to face the lumen of the pentameric oligomer using site-specific infrared dichroism of the isotopically labeled TM domain reconstituted in model lipid bilayers . In the NMR based model of the full length protein (Fig. 2), although His22 adopts a lumenal orientation, the second histidine, His51, appears in an extramembrane location, at the tip of the C-terminal extended loop (Fig. 2A), which is difficult to reconcile with an activation role based on His protonation. Thus, it is possible that the structure of this C-terminal domain in detergent micelles, used for the NMR experiment, does not represent accurately the structure of SH protein in lipid bilayers, where we obtained the patch clamp data. Nevertheless, the pH-activated channel activity observed, and the histidine-less inactive mutant strongly suggests that protonation of histidines may be involved in channel activity. Indeed, the presence of a lumenal histidine sidechain is reminiscent of the one found in the TM domain of the influenza A AM2 proton channel, which is also activated at low pH via histidine protonation .
Despite the similarities between AM2 and SH protein, we have been unable to observe strong proton channel activity of SH protein
2.2. E protein in coronaviruses
SARS produced a near pandemic in 2003, with 8,096 infected cases and 774 deaths worldwide (fatality rate of 9.6%). Mortality was 6% for those aged 25-44, 15 % for the 45-64 group and >50% for those over 65 (
Protective efficacy of candidate vaccines against coronaviruses in humans has been mainly studied in animals so far, and only few vaccines have entered Phase 1 human trials . Ribavirin , interferons , unconventional agents [81-83] and non-steroidal anti-inflammatory agents  have shown activity against SARS-CoV and HCoV-229E, but there is no data from animal studies or clinical trials . Studies of antiviral therapy against coronaviruses other than SARS-CoV have been scarce;
In addition to the genes involved in viral RNA replication and transcription, other essential genes in coronaviruses encode the common viral structural proteins, S (spike), E (envelope), M (membrane) and N (nucleocapsid). Of these, S, E, and M are incorporated into the virion lipidic envelope, and S protein is involved in fusion with host membranes during entry into cells. The M protein is the most abundant constituent of coronaviruses and gives the virion envelopes their shape; the E protein is only a minor constituent of the virion but is abundantly expressed inside the infected cell [88-90].
The E protein in SARS-CoV is the shortest, with only 76 amino acids, whereas that of IBV E is one of the longest (109 amino acids). E protein sequences are extremely divergent in their sequence, but the same general architecture is found in all of them: a short hydrophilic N-terminus (8–12 residues), an N-terminal TM domain (21–29 residues) followed by a cluster of 2-3 cysteines which are likely to be palmitoylated, and finally a less hydrophobic C-terminal tail (39–76 residues). Prediction of TM domains of representatives of coronavirus E proteins from several species using a hidden Markov model (e.g., http://phobius.sbc.su.se/)  shows that they have at least one α-helical TM domain. In some cases a second TM domain is also predicted, e.g., in IBV E and MHV E (Fig 3). However, in none of these coronavirus E proteins this second putative TM has a predicted α-helical conformation. Instead, a β-coil-β motif appears to predominate in that part of the sequence, with a totally conserved Pro residue in a central position (‘P’ in Fig. 3).
The results of these experiments should be interpreted with caution, especially comparing data from transfected cells and infected cells. Equally, the possible lack of accessibility to antibodies of parts of the protein plays a part. Indeed, as discussed elsewhere , in the case of IBV E, if the entire N-terminal region of IBV E protein was buried within the intracellular membrane, it would have remained inaccessible to the antibodies used. Several coexisting forms may exist for E proteins, which would have different roles in the life cycle of the virus.
The factors that would favour one topology over another are unknown, but one possible candidate is palmitoylation. Indeed, E proteins of SARS , IBV  and MHV  are palmitoylated at one or more cysteines. This modification is likely to have structural and functional consequences, because removal of the cysteines in MHV E resulted in deformed viruses [100, 101]. Experimental determination of the topology of these E protein homologs – with or without palmitoylation – in model membranes or membrane-like detergents is critical to understand the function of the envelope protein in coronavirus biology. Unfortunately, these detailed structural studies are still not available.
The importance of the correct topology in E proteins may be highlighted by a recent study  that showed that E protein in MHV could be replaced by some heterologous E proteins. The MHV virus became viable when the replacement was from groups 2, i.e., β-coronaviruses (SARS-CoV E) and 3, i.e., γ-coronaviruses (IBV E), but not when TGEV E (group 1, or α-coronaviruses) was used. This discrimination may have to do with topology considerations, because the contribution of E proteins to the formation of viral particles in coronaviruses could be provided by a broad range of sequences, and not by specific interactions.
In other coronaviruses, it has been found that E protein is involved in viral morphogenesis, e.g., co-expression of M and E is sufficient for formation and release of virus-like particles (VLP) in the host cell [93, 111-115]. Also, mutations in the extramembrane domain of E protein were shown to impair viral assembly and maturation in MHV , probably due to a defective interaction with M protein. In TGEV, the absence of E protein resulted in a blockade of virus trafficking in the secretory pathway, and the prevention of virus maturation [117, 118].
Previous reports have studied the oligomerization of coronavirus E proteins. However, results were not conclusive, partly because these experiments were performed in SDS, a harsh detergent that leads to monomers or to non-specific aggregates. For example, SARS-CoV E oligomerization has been studied in Western Blots after SDS-PAGE and labeling with polyclonal antibodies , antibodies against a hemagglutinin-derived C-terminal tag , or using non purified or truncated synthetic E proteins [124, 125]. In the latter approach, a predominanly monomeric form was observed in SDS. In our hands, synthetic SARS-CoV E also produced in SDS mostly monomers, and a minor fraction of dimers (unpublished observations), but several oligomers were observed for the recombinant form (Fig. 4, lane WT). The differences between synthetic and recombinant E protein may be due to unwanted side reactions that take place during synthesis. Addition of DTT (Fig. 4B) produces bands compatible with monomers and trimers, whereas cysteine-less mutants only produced monomers. Thus, the three cysteines in SARS-CoV E seem to participate in some inter-monomeric contacts. Indeed, sedimentation data for SARS-CoV E could only be fitted after addition of reductant  in the case of SARS-CoV E. Differences between absence and presence of reductant were observed even when only one cysteine was available (Fig. 4B), therefore these disulfide bonds may not be specific. Changes in hydrophobicity and local secondary structure seem to play a major role in the results observed. Further, disulfide bonds are not necessary to form pentamers; sedimentation equilibrium of full-length SARS-CoV E or IBV E in C14SB detergent and in the presence of reducing agent produced best fit to a monomer-pentamer equilibrium model , similar to what has been observed for the TM region alone .
The likely orientation of these cysteine residues relative the pentameric bundle can be determined on the basis of the available structure formed by synthetic TM8-38 . That structure did not include any of the three cysteines of SARS-CoV E, but if the structural model is prolonged by two turns (Fig. 5), the three cysteines are seen oriented either towards the lumen of the channel or inter-helically.
The juxtamembrane cysteines in coronavirus envelope proteins are well conserved, and have been found to be crucial in the coronavirus cycle. For example, in MHV E, removal of the cysteines resulted in deformed viruses [99-101]. Using the full length infectious clone , double- and triple-mutants to alanine produced smaller plaques and decreased virus yields. Single-substitution mutants, in contrast, did not produce anomalous growth, whereas replacement of all three cysteines resulted in crippled virus with significantly reduced yields. In these reports, these effects were attributed to the absence of palmitoylation sites, which may direct E proteins towards lipid rafts . E proteins of SARS , IBV  and MHV  have been shown to be palmitoylated at one or more cysteines. It is possible that an additional role of palmitoylation is to drag the C-terminal tail of E proteins towards the membrane and trigger a conformational change.
Similar experiments with fragments 46-60 and 59-76 (Fig. 6, c-d) showed that 46-60 forms β-sheets resistant to H/D exchange. Indeed, this latter peptide showed limited solubility, similar to the ‘parent’ peptide 36-76. Further, its amide I spectrum in DMPC displayed the features of antiparallel β-sheet, with bands at 1635 cm-1 and 1685 cm-1 (Fig. 6c) and showed no H/D exchange in the amide II region (Fig. 6c, star). In contrast, fragment 61-76 is predicted to form random coil (Fig. 6a), and should show complete H/D exchange. Indeed, the hydrophilic fragment 59–76 dissolved readily in water (>5 mg/ml), produced an amide I spectrum in DMPC consistent with random structure (Fig. 6d), with a broad amide I band at 1645 cm-1, and showed complete H/D exchange at the amide II region (star).
The two folding domains observed in the C-terminal domain of SARS-CoV E are reminiscent of the two separate domains reported for the amyloid peptide , where fragment 34–42 has limited solubility and adopts antiparallel β-sheet structure, and fragment 26–33 is more soluble in water, and has a disordered conformation. Thus, we tested if peptide (36-76) can form amyloid-like fibrils. The aggregate obtained after drying this peptide from acetonitrile showed intense X-ray reflections at ~4.8 Å and ~10.8 Å (Fig. 6f), which correspond to the distances between hydrogen bonded peptide backbones and β–pleated sheets, respectively, characteristically found in Alzheimer disease amyloid plaque cores . This peptide was monomeric in SDS. Based on the above results, a topological model for SARS-CoV E can be proposed, with one α-helical TM domain and a C-terminal β-hairpin (Fig. 6g). We have reported previously that SARS-CoV E secondary structure in lipid bilayers is predominantly α-helical , in contrast with the results shown in Fig. 6e. However, we have found that the secondary structure of E protein is strongly dependent on the reconstitution conditions. In our previous report , the protein was presolubilized in hexafluoroisopropanol, an α-helix inducer, whereas in Fig. 6e pre-solubilization was done in methanol. Thus, the β-hairpin prediction for the residues around the highly conserved residue P54, may be correct only in certain experimental conditions. The dual conformation, α-helical and β-hairpin, conformation proposed here is reminiscent of the proposed dual topology of a similar β-hairpin with central conserved Pro residue found in stomatin. In that case, secondary structure changed to α-helix when Pro was mutated to Ser .
Examination of the peptide (36-76) precipitate by electron microscopy (Fig. 7A) revealed a protofibrillar morphology [139-141]. These structures were not observed in the control specimen prepared in the absence of peptide (Fig. 7B). The fibrils form an ordered mesh structure characterized by straight sections intervened by bends. The fibril width was 7, 8 and 10 nm, consistent with reports of other filaments derived from β-sheet structures [141-143]. The length of the straight sections was rather homogenous, with most measurements falling between 20-40 nm and with an average value of 32 nm.
The formation of fibrils is anticipated by the residue composition in the region around the conserved proline (P54). For example, from the 17 residues in the stretch I46 to V62, 9 residues are either V, I or Y. Amino acids with β-branched side chains, e.g. valine and isoleucine, or bulky residues, have been shown previously to disfavor α-helical conformation, and to pack efficiently along the surface of a β-sheet [144, 145]. Accordingly, a series of hexapeptides containing similar motifs (e.g., VxVx) have been shown to be good amyloid-forming peptides . We speculate that changing some of these residues to non-branched, for example from V to L, would abolish the ability of SARS-CoV E to form fibers, and possibly attenuate the observed cytopathological effects of SARS-CoV E in cells. Indeed, a similar strategy led to disruption of Golgi targeting in SARS-CoV E .
In addition, a sequence of ordered fragments (α-helices or strands) flanking a disordered or turn loop, with Pro at its center, has been described for several fusion peptides, e.g., in EnvA of the Avian sarcoma/leukosis virus subtype A (ASLV-A) , Ebola virus GP  and mouse or macaque fertilin α (ADAM 1) , which suggests that this part of SARS-CoV E is analogous to an internal fusion peptide. This motif has also been observed in a cis-proline turn  linking two β-hairpin strands in the structure of an HIV-1IIIB V3 peptide. It was found by mutagenesis of the fusion peptide of Env in ASLV-A, that proline, or a residue of similar intermediate hydrophobicity, are part of an accessible loop and was needed for initial interactions of fusion peptides with target membranes.
Amyloid fiber formation has been reported for fragments of many non pathogenic proteins , and they have been found in a variety of proteins which are not associated with disease [152, 153]. Therefore, this finding may not have relevance for the toxicity of the virus. Nevertheless, this possibility cannot be discarded in view of other roles of similar semen-derived fibers in HIV viral entry which dramatically enhance HIV infection . A more likely possibility, however, is that this conformational plasticity is needed during membrane fusion; a transition form a α-helical conformation to an antiparallel β-structure, with Pro as a hinge, could drive membrane fusion by pulling the two membranes in close apposition.
Viroporins constitute important components of viruses, and we are just beginning to understand what is their biological role during the viral life cycle. One of the main problems in their