The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins

Abstract

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

Re-annotation of SARS-CoV-2 proteins using an HHpred-based approach opens new opportunities for a better understanding of this virus

“…From this point of view, our prediction is therefore in line with the work of (Kern et al, 2021). However, it is worth mentioning that a recent work challenges the results of both (Kern et al, 2021) and (Lu et al, 2006): (Miller et al, 2023) provide evidence suggesting that while a narrow cavity is detected in the SARS-CoV-2 ORF3a transmembrane region, it likely does not represent a functional ion-conducting pore (the same holds true for SARS-CoV-1 ORF3a).…”

Tetherin antagonism by SARS‐CoV‐2 ORF3a and spike protein enhances virus release

“…Expression of ORF3a impairs endolysosomal acidification (Ghosh et al, 2020) and promotes lysosome fusion with the plasma membrane (Chen et al, 2021). ORF3a has been proposed to dimerise and form an ion channel (Kern et al, 2021;Zhang et al, 2022a), although recent structural and functional experiments suggest that ORF3a is not an ion channel and instead sequestrates VPS39, impairing fusion of late endosomes with lysosomes (Miao et al, 2021;Miller et al, 2023).…”

“…Ó 2023 The Authors EMBO reports 24: e57224 | 2023 SARS-CoV-2 ORF3a is an accessory protein that localises to and perturbs endosomes and lysosomes (Miao et al, 2021). It may do so by acting either as a viroporin (Zhang et al, 2022a) or by interacting with, and possibly interfering with the function of VPS39, a component of the HOPS complex which facilitates tethering of late endosomes or autophagosomes with lysosomes (Miao et al, 2021;Miller et al, 2023). Given ORF3a likely impairs lysosome function, the observed increased presence of tetherin puncta following ORF3a expression may be due to decreased lysosomal degradation.…”

To be or not to be an ion channel: cryo-EM structures have a say

“…The authors also performed comprehensive electrophysiological experiments including whole-cell and endolysosomal patch clamp in HEK293 cells, and recordings in Xenopus oocytes and reconstituted proteoliposomes, but none of these attempts have obtained evidence supporting the channel activity of ORF3a. The large currents from reconstituted proteoliposomes are generally resulted from membrane leakiness and/or contamination of endogenous channel proteins in the purified sample (Miller et al , 2023). Different from ORF3a, other proposed SARS-COV-2 viroporins, including protein E, ORF7b and ORF10, are predicted to possess only one or no transmembrane segment (Fig.…”