PT  - JOURNAL ARTICLE
AU  - Alexandria N. Miller
AU  - Patrick R. Houlihan
AU  - Ella Matamala
AU  - Deny Cabezas-Bratesco
AU  - Gi Young Lee
AU  - Ben Cristofori-Armstrong
AU  - Tanya L. Dilan
AU  - Silvia Sanchez-Martinez
AU  - Doreen Matthies
AU  - Rui Yan
AU  - Zhiheng Yu
AU  - Dejian Ren
AU  - Sebastian E. Brauchi
AU  - David E. Clapham
TI  - The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins
AID  - 10.1101/2022.09.02.506428
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.09.02.506428
4099  - http://biorxiv.org/content/early/2022/09/03/2022.09.02.506428.short
4100  - http://biorxiv.org/content/early/2022/09/03/2022.09.02.506428.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.