Channel-Inactivating Mutations and Their Revertant Mutants in the Envelope Protein of Infectious Bronchitis Virus

Abstract

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.

Coronavirus envelope protein: current knowledge

“…This suggests that while some of these mutations appear to merely restore the loss of ion channel activity, it is not entirely inconceivable that revertant viruses would acquire gain of function mutations that can render it more virulent [77]. Similar results were recently reported for IBV E TMD residues analogous to N15A and V25F (T16A and A26F) [222]. It is interesting to note that in both cases SARS-CoV E and IBV E followed a similar trend in their reversion: mutations at N15A and T16A both reverted by substitution of a single residue, whereas mutations at V25F and A26F produced revertants by acquisition of multiple residues.…”

Regulation of the ER Stress Response by the Ion Channel Activity of the Infectious Bronchitis Coronavirus Envelope Protein Modulates Virion Release, Apoptosis, Viral Fitness, and Pathogenesis

“…CoV E protein is a small structural protein that serves critical functions in the morphogenesis and release of mature virions (DeDiego et al, 2007;To et al, 2017), and its recently characterized IC activity has been implicated in modulating viral pathogenesis . In this study, we have constructed five rIBVs: the parental rIBV-p65, two rIBVs harboring IC-inactivating mutations in the E protein (rT16A and rA26F), and two putative revertants harboring one additional mutation in the TMD of E protein (rT16A/A26V and rA26F/F14N, respectively).…”

Post-Translational Modifications of Coronavirus Proteins: Roles and Function

“…into the envelope and provides structural support to the virion. The E protein is a small, integral membrane protein present at a low amount in the virion, but it plays an essential role during virion assembly and release [11,12]. Inside the envelope, the helically symmetric nucleocapsid is comprised of the RNA genome closely associated with the N protein in a beads-on-a-string fashion.…”

“…Current evidence suggests E protein as a type I transmembrane protein with a short N-terminal ectodomain and a C-terminal endodomain ( Figure 3A), but alternative membrane topologies have also been proposed [79,80]. Biophysical studies show that some coronavirus E proteins can form pentameric structures exhibiting ion channel activity [12,81]. The E protein is reported to be modified by glycosylation and palmitoylation ( Figure 3B & Table 1) [80,82].…”