Search

It has been shown previously in the severe acute respiratory syndrome coronavirus (SARS-CoV) that two point mutations, N15A and V25F, in the transmembrane domain (TMD) of the envelope (E) protein abolished channel activity and led to in vivo attenuation. Pathogenicity was recovered in mutants that also regained E protein channel activity. In particular, V25F was rapidly compensated by changes at multiple V25F-facing TMD residues located on a neighboring monomer, consistent with a recovery of oligomerization. Here, we show using infected cells that the same mutations, T16A and A26F, in the gamma-CoV infectious bronchitis virus (IBV) lead to, in principle, similar results. However, IBV E A26F did not abolish oligomer formation and was compensated by mutations at N- and C-terminal extramembrane domains (EMDs). The C-terminal EMD mutations clustered along an insertion sequence specific to gamma-CoVs. Nuclear magnetic resonance data are consistent with the presence of only one TMD in IBV E, suggesting that recovery of channel activity and fitness in these IBV E revertant mutants is through an allosteric interaction between EMDs and TMD. The present results are important for the development of IBV live attenuated vaccines when channel-inactivating mutations are introduced in the E protein. IMPORTANCE The ion channel activity of SARS-CoV E protein is a determinant of virulence, and abolishment of channel activity leads to viral attenuation. E deletion may be a strategy for generating live attenuated vaccines but can trigger undesirable compensatory mechanisms through modifications of other viral proteins to regain virulence. Therefore, a more suitable approach may be to introduce small but critical attenuating mutations. For this, the stability of attenuating mutations should be examined to understand the mechanisms of reversion. Here, we show that channel-inactivating mutations of the avian infectious bronchitis virus E protein introduced in a recombinant virus system are deficient in viral release and fitness and that revertant mutations also restored channel activity. Unexpectedly, most of the revertant mutations appeared at extramembrane domains, particularly along an insertion specific for gammacoronaviruses. Our structural data propose a single transmembrane domain in IBV E, suggesting an allosteric interaction between extramembrane and transmembrane domains. [ABSTRACT FROM AUTHOR]

A family of small polypeptides in many virus types associate to form oligomers and have channel activity. These proteins have been referred to as viroporins or virochannels and are increasingly recognized as important virulence factors and potential drug targets. In this review, we focus on two of the viroporins that have been studied in more detail from a structural and functional point of view. One is the 76-residue envelope (E) protein found in coronaviruses (CoVs) that causes the severe acute respiratory syndrome (SARS). The other is the 65-residue small hydrophobic (SH) protein found in a paramyxovirus, the respiratory syncytial virus (RSV). RSV SH and SARS-CoV E proteins are short polypeptides with a single transmembrane domain. In both cases, the presence of the viroporin has a protective effect on cells, preventing early apoptosis, but it leads to increased virulence in infected animal models. Both viroporins form homopentameric oligomers that show channel activity with no or low selectivity. The role of channel activity is still unclear, but associations have been made to facilitation of the egress of the virus by modification of the secretory pathway, and contributions to inflammation. SARS-CoV E protein has a cytoplasmically oriented C-terminus and a lumenal N-terminus, whereas the opposite orientation is found in RSV SH protein. Despite this opposite topology, nuclear magnetic resonance (NMR)-based structural models of these two channels show a similar champagne flute shape, with the wider opening facing the cytoplasmic side. Good channel inhibitors are lacking, but those found seem to have a preference for the narrow end of the channel. Availability of good inhibitors will help reveal the specific role of these channels in the life cycle of these viruses. [ABSTRACT FROM AUTHOR]

Coronavirus envelope (CoV E) proteins are ~100-residue polypeptides with at least one channel-forming α-helical transmembrane (TM) domain. The extramembrane C-terminal tail contains a completely conserved proline, at the center of a predicted β-coil-β motif. This hydrophobic motif has been reported to constitute a Golgi-targeting signal or a second TM domain. However, no structural data for this or other extramembrane domains in CoV E proteins is available. Herein, we show that the E protein in the severe acute respiratory syndrome virus has only one TM domain in micelles, whereas the predicted β-coil-β motif forms a short membrane-bound α-helix connected by a disordered loop to the TM domain. However, complementary results suggest that this motif is potentially poised for conformational change or in dynamic exchange with other conformations. [ABSTRACT FROM AUTHOR]