SARS-CoV-2 E and 3a proteins are inducers of pannexin currents

Abstract

Controversial reports have suggested that SARS-CoV E and 3a proteins may be viroporins that conduct currents through the plasma membrane of the infected cells. If true, these proteins would represent accessible targets for the development of new antiviral drugs by using high-throughput patch-clamp techniques. Here we aimed at better characterizing the cell responses induced by E or 3a protein with a particular focus on the ion conductances measured at the cell surface. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly-acquired round shape, tending to detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this hypothesis by using flow cytometry, in agreement with earlier reports on other cell types. In adhering cells expressing E or 3a protein, whole-cell currents were in fact not different from the control condition indicating that E and 3a proteins are not plasma membrane viroporins. In contrast, recording currents on detached cells uncovered outwardly-rectifying currents, much larger than those observed in control. The current characteristics are reminiscent of what was previously observed in cells expressing SARS-CoV-1 E or 3a proteins. Herein, we illustrate for the first time that carbenoxolone blocks these outward currents suggesting that they are conducted by pannexin channels, mostly likely activated by cell morphology change and/or cell death. Alongside we also demonstrate that truncation of the C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent pannexin currents suggesting distinct pathways for cell death and pannexin currents induced by E and 3a proteins. We conclude that SARS-CoV-2 E and 3a proteins are not acting as viroporins expressed at the plasma membrane.

SARS-CoV-2 ORF3a-Mediated NF-κB Activation Is Not Dependent on TRAF-Binding Sequence

“…We note that the scope of our current study is limited to NF-κB activation by SARS-CoV-2 ORF3a. ORF3a is reported to affect multiple signaling pathways including apoptosis, autophagy, and inflammasomes, in addition to modulating cellular membrane structures and forming cation channels [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. This multifunctional capacity is not unique to SARS-CoV-2 ORF3a but has been shown for other coronavirus accessory proteins, such as ORF3 from porcine epidemic diarrhea virus (PEDV), another proposed viroporin that shares many structural and functional features with ORF3a.…”

“…ORF3a dimers have also been thought to form a cation channel [8,9,16,25]. However, recent studies have challenged this hypothesis, noting that the proposed pore is not suitable for cation permeation based on the cryo-EM structures [10,26]. Instead, the previously observed ion conductance may be due to ORF3a-induced pannexin channel activities [26].…”