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Infectious Bronchitis Virus | 141

Figure 5.9 Linear and folded representations of coronavirus envelop (E) protein. Top Important residues for the infectious bronchitis virus
(IBV) E protein are T and A (ion channel activity). The C-terminal RDKLYS contains an endoplasmic reticulum (ER) retention signal. Below
16 26
The positions of T and A are shown (boxed). TM, transmembrane domain.
16 26
the E gene deleted have been successfully generated for MHV abolishes the channel conductance (To et al., 2017). Interest-
and SARS-CoV, although the mutants formed severely crippled ingly, overexpression of IBV E protein, but not the T16A mutant,
virions with significantly reduced titres (Kuo and Masters, 2003; disrupted the intracellular trafficking of VSV-G protein and the
DeDiego et al., 2007). However, recombinant IBV with the E morphology of the Golgi complex (Ruch and Machamer, 2012).
gene deleted could not be recovered, indicating that E protein Later biochemical analysis has shown that in both infected cells
plays a critical role during IBV replication (unpublished data). In and virions, the IBV E protein is present in two distinct pools: a
fact, simply swapping the HD domain of IBV E protein with the high molecular weight (HMW) pool and a low molecular weight
transmembrane domain of VSV-G resulted in ≈ 200-fold reduc- (LMW) pool (Westerbeck and Machamer, 2015). The virion-
tion of virion release into the supernatant (Machamer and Youn, associated IBV E was mainly in the HMW pool and formed
2006). homo-oligomers. Interestingly, at steady state, the T16A protein
Biophysical and computational studies have supported a was nearly exclusively in the HMW pool, while the A26F protein
model that five molecules of SARS-CoV E protein form a homo- was enriched in the LMW pool (Westerbeck and Machamer,
pentameric α-helical bundle, with the hydrophobic domains 2015). Consistently, the T16A mutant could support the produc-
embedded in the lipid bilayer, forming a voltage-independent tion of virus like particles (VLPs) similar to the wild type IBV E
ion channel (Torres et al., 2006; Verdiá-Báguena et al., 2012). protein, whereas the A26F mutant did not support VLP produc-
Ion channel activity of SARS-CoV E protein could be readily tion (Westerbeck and Machamer, 2015).
determined in vitro, and its overexpression also altered mem- To better understand the importance of the ion channel activ-
brane permeability in both E. coli and mammalian cells (Liao ity of E protein during IBV replication, a recombinant IBV with
et al., 2004, 2006). Two mutations within the HD of SARS- either T16A or A26F mutation was generated (To et al., 2017).
CoV E protein, namely N15A and V25F, have been shown to Both mutant rIBVs could be recovered, although A26F formed
completely abolish the ion channel activity (Verdiá-Báguena et smaller plaques compared with wild type. The two ion channel
al., 2012). mutants were very similar to wild type IBV in terms of genome
It is very likely that IBV E protein adopts similar membrane replication and transcription, structural protein synthesis and
topology and forms a homopentamer as the SARS-CoV E protein virion assembly. However, the release of mature virion to the
(Fig. 5.9). Biophysical analysis has shown that IBV E protein culture supernatant was significantly reduced in the mutants,
also exhibited ion channel activity and either of the correspond- indicating that ion channel activity is required for efficient release
ing mutations in the HD domain, T16A and A26F, completely of infectious virus particles (To et al., 2017).