The β-coronavirus, Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) has among the largest genomes of any RNA virus (Bar-On et al., 2020; Hartenian et al., 2020), encoding for 29 proteins. The majority of these proteins are ‘Non-Structural Proteins’ (NSPs) that mediate viral RNA replication, while the ‘structural proteins’ are components of the virion (Bar-On et al., 2020; Hartenian et al., 2020). The third group of functionally enigmatic ’accessory’ proteins likely bolster SARS-CoV-2 replication or facilitate evasion from the host’s innate immune system. One of these proteins is Open reading frame 3a (Orf3a), a membrane protein that is annotated as a putative viroporin based on previous work and its similarity to SARS-CoV-1, also claimed to be a viroporin (Kern et al., 2021; Toft-Bertelsen et al., 2021; Lu et al., 2006; Chan et al., 2009). In this paper, we determine the structures of SARS-CoV-2 and SARS-CoV-1 Orf3a, measure their functions, and examine the differences between the CoV-2 and CoV-1 proteins.

Viroporins are viral membrane proteins that commonly form weakly- or non-selective oligomeric holes in surface plasma membranes or intracellular organellar membranes (Nieva et al., 2012; Scott and Griffin, 2015). SARS-CoV-1 Orf3a was postulated to be a viroporin based on its membrane localization, oligomerization, and C-terminal sequence similarities to a calcium ATPase from Plasmodium falciparum and an outer-membrane porin from Shewanella oneidensis (Lu et al., 2006; Yu et al., 2004). Recording K⁺-selective currents from SARS-CoV-1 Orf3a-expressing Xenopus laevis oocytes were interpreted to mean that SARS-CoV-1 Orf3a was a viroporin (Lu et al., 2006). This current was inhibited by 5–50 µM emodin, a natural compound suggested as a potential SARS-CoV-1 antiviral (Lu et al., 2006; Schwarz et al., 2011), and by 10 mM BaCl₂, a nonspecific K⁺ channel blocker. Whole-cell patch-clamp of SARS-CoV-1 expressing HEK293 cells exhibited non-selective ion currents blocked by BaCl₂ that were also attributed to SARS-CoV-1 Orf3a (Chan et al., 2009). Although these data were used to support the SARS-CoV-1 Orf3a viroporin hypothesis, further work to identify its pore-lining residues is lacking (Lu et al., 2006; Chan et al., 2009). One study pursued this question by testing SARS-CoV-1 Orf3a pore mutants in artificial bilayers (Castaño-Rodriguez et al., 2018). However, this method is prone to background channel contamination and the validity of this observation has been recently questioned (Accardi et al., 2004; McClenaghan et al., 2020).

Comparison of viral infections between wild-type and Orf3a-deficient SARS-CoV-1 strains demonstrates that Orf3a promotes host cell death and causes intracellular vesicle formation and Golgi fragmentation (Freundt et al., 2010). In addition, during SARS-CoV-1 infection, Orf3a stimulates the production of mature IL-1β, a marker of NLRP3 inflammasome activation (Siu et al., 2019). Although viroporins from other viruses have been shown to trigger apoptosis or innate immune cell activation, many other viral hijacking mechanisms can produce these phenotypes (Nieva et al., 2012; Scott and Griffin, 2015). The lack of compelling evidence connecting the contributions of Orf3a to SARS-CoV-1 pathogenesis with its purported viroporin activity raises the question of whether its channel function is physiologically required and if SARS-CoV-1 Orf3a is a bona fide viroporin.

Given its similarity to the SARS-CoV-1 homolog, we asked if SARS-CoV-2 Orf3a is a viroporin. While pursuing this, several groups reported conflicting findings that support or oppose the SARS-CoV-2 Orf3a viroporin hypothesis (Kern et al., 2021; Toft-Bertelsen et al., 2021; Grant and Lester, 2021). We reasoned that the discrepancy could be due to common experimental problems that arise when characterizing putative ion channels, leading to its misidentification as a viroporin (Accardi et al., 2004; Harrison et al., 2022; Niu et al., 2021). Additionally, SARS-CoV-2 Orf3a viroporin activity has not been studied in mammalian cells, which represents a more native environment for functional studies (Kern et al., 2021; Toft-Bertelsen et al., 2021; Grant and Lester, 2021). We performed a comprehensive structural and functional investigation of SARS-CoV-2 Orf3a. We surveyed the subcellular localization of overexpressed SARS-CoV-2 Orf3a in HEK293 cells and observed co-localization with markers of the plasma membrane and the endocytic pathway, as previously reported (Ghosh et al., 2020; Gordon et al., 2020a; Zhang et al., 2020; Miao et al., 2021). We then made extensive efforts to record SARS-CoV-2 Orf3a attributable cation currents at the plasma membrane and in endolysosomes of HEK293 cells. We also attempted to measure SARS-CoV-2 Orf3a currents at the plasma membrane of A549 lung alveolar cells (a model of alveolar type II pneumocytes that are a target host cell of SARS-CoV-2 infection), in Xenopus oocytes, and in a reconstituted system. In all cell lines and reconstituted systems tested, we did not measure currents attributable to SARS-CoV-2 Orf3a. We explored this further by resolving three high-resolution cryo-EM structures of SARS-CoV-2 Orf3a under different conditions, varying the lipid composition and the scaffold protein used for nanodisc assembly. All structures were captured in the same conformational state, displaying a constriction within the transmembrane region and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We were also unable to reproduce the published SARS-CoV-1 Orf3a recordings from Xenopus oocytes and HEK293 cells. Finally, we show that the SARS-CoV-1 Orf3a cryo-EM structure mirrors the overall architecture and structural features seen in the SARS-CoV-2 homolog. From our data, we conclude that SARS-CoV-1 and SARS-CoV-2 Orf3a are not viroporins.

What is the function of SARS-CoV-2 Orf3a and how may it contribute to SARS-CoV-2 pathogenicity? We observe Rab7 enrichment, a marker for late endosomes, upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with the host protein, VPS39. In contrast, SARS-CoV-1 Orf3a does not cause the same cellular phenotype and does not interact with VPS39. We identified an unstructured, cytosolic loop unique to SARS-CoV-2 Orf3a that contributes to its interaction with VPS39. Our data is in agreement with previous work showing that SARS-CoV-2 Orf3a binds VPS39, a HOPS complex tethering protein involved in autophagosome and late endosome fusion with lysosomes (Miao et al., 2021; Chen et al., 2021; Qu et al., 2021; Balderhaar and Ungermann, 2013). Disruption of HOPS complex activity may be a mechanism to promote SARS-CoV-2 viral egress, an exit strategy recently proposed for β-coronaviruses (Ghosh et al., 2020; Chen et al., 2021).

Viroporins are small viral membrane proteins that often form weakly- or non-selective pores (Nieva et al., 2012; Scott and Griffin, 2015). They are widely distributed among different viral families and, accordingly, their contributions to viral survival range widely from assisting with viral propagation to participating in host immune evasion (Nieva et al., 2012; Scott and Griffin, 2015). Four proteins encoded by SARS-CoV-1 have been proposed to function as viroporins - E protein, Orf3a, Orf8a and Orf10 (Lu et al., 2006; Harrison et al., 2022; Liao et al., 2004; Chen et al., 2011). To demonstrate that a novel protein unequivocally forms a viroporin requires the identification of its pore-lining residues. This is ideally done by introducing a pore-lining cysteine that can be modified by a methanethiosulfonate reagent, which when added, partially blocks the pore or alters ion channel selectivity (Newell and Czajkowski, 2007; Frillingos et al., 1998). This has yet to be demonstrated for SARS-CoV-1 or SARS-CoV-2 Orf3a.

We asked if Orf3a from SARS-CoV-2 is a bona fide viroporin and took a comprehensive approach to investigate its function in several mammalian cell lines. We first assessed subcellular localization of SARS-CoV-2 Orf3a to determine where it may be functioning in mammalian cells and observed its enrichment at the PM and in the endocytic pathway. We then performed whole-cell patch-clamp in HEK293 and A549 cells, and endolysosomal patch-clamp from HEK293 cells, in numerous cationic conditions. We also tested SARS-CoV-2 Orf3a at the PM of Xenopus oocytes and in reconstituted systems using purified SARS-CoV-2 Orf3a. We were unable to record currents that we can attribute to SARS-CoV-2 Orf3a.

While performing these studies, several other groups published electrophysiology data to either support or oppose the SARS-CoV-2 Orf3a viroporin hypothesis (Kern et al., 2021; Toft-Bertelsen et al., 2021; Grant and Lester, 2021). Our results from Xenopus oocytes are consistent with Grant and Lester, 2021. We propose that the reported SARS-CoV-2 Orf3a current observed by Toft-Bertelsen et al. could be due to a technical artifact such as pipette leak or contributed by endogenous channels (Toft-Bertelsen et al., 2021; Harrison et al., 2022; Ackerman et al., 1994). Similar to Kern et al., we were able to measure multiple large single channel-like conductances from vesicle-reconstituted SARS-CoV-2 Orf3a in K⁺, Na⁺, and Ca²⁺ conditions using an atypically high protein to lipid ratio (1:10, wt:wt) (Kern et al., 2021; Miller, 1986). These channel-like conductances were not present in samples reconstituted using a standard protein to lipid mixture of 1:100 (wt:wt) (Miller, 1986). The lack of macroscopic currents recorded from vesicles containing high protein amounts was concerning since we estimate that ~1,000 putative channels/μm² should be present (Goldberg and Miller, 1991; Maduke et al., 1999). We suspect that these channel-like conductances reflect spontaneous leak due to the high protein amounts in the sample, or possibly a contaminating channel, such as VDAC1 (Niu et al., 2021). Using several flux assays, we did not observe K⁺ or Cl^- flux from any of our reconstituted conditions, further indicating that the channel-like conductances are likely spurious.

To attempt to capture SARS-CoV-2 Orf3a in different conformational states, we determined three cryo-EM structures of SARS-CoV-2 Orf3a in lipid environments mimicking the PM and LE/Lyso and in a nanodisc containing a different membrane scaffold protein. We observe one nearly identical state in all SARS-CoV-2 Orf3a sample preparations, consistent with the published cryo-EM structure of SARS-CoV-2 Orf3a, determined at 2.1 Å resolution (global RMSD 0.38 Å between the SARS-CoV-2 Orf3a PM MSP1D1 nanodisc and published SARS-CoV-2 Orf3a structures, Figure 3—figure supplement 3K; Kern et al., 2021). We do not observe a membrane-spanning pore wide enough to permit the movement of dehydrated cations. Furthermore, the positive electrostatic potential within the aqueous vestibule would not favor cation permeation.

We also attempted to reproduce published data by investigating SARS-CoV-1 Orf3a viroporin activity in both HEK293 cells and Xenopus oocytes (Lu et al., 2006; Chan et al., 2009). We were unable to record cationic currents attributable to SARS-CoV-1 Orf3a in either system, yet we observed its localization at the PM. Similarly, we were unable to observe K⁺ flux from vesicle-reconstituted SARS-CoV-1 Orf3a. We also determined a cryo-EM structure of SARS-CoV-1 Orf3a in an LE/Lyso MSP1D1-containing nanodisc and found its overall architecture indistinguishable from the SARS-CoV-2 homolog. From our comprehensive evaluation of both SARS-CoV-1 and SARS-CoV-2 Orf3a in multiple cell lines and reconstituted systems, and the conservation between the cryo-EM structures, we conclude that SARS-CoV-1 and SARS-CoV-2 Orf3a proteins are not viroporins.

Although a wide, membrane-spanning pore is not evident from the SARS-CoV-1 and SARS-CoV-2 Orf3a structures, we do observe density in our cryo-EM maps that we attribute to lipids. By comparing our four cryo-EM maps – three SARS-CoV-2 Orf3a samples reconstituted into nanodiscs containing PM- or LE/lyso-like lipid compositions and either MSP1D1 or Saposin A protein scaffolds, and one SARS-CoV-1 Orf3a sample reconstituted into a MSP1D1-containing nanodisc in a LE/Lyso-like environment – we detect two independent lipid binding sites likely occupied by DOPE. The cytosolic orientation of the two Orf3a fenestrations further supports the assignment of PE lipids since they are enriched in the inner leaflet of the PM and likely LE/Lyso (van Meer et al., 2008). We reason that Orf3a is not engaged with both molecules simultaneously due to steric clashes with R122 and between the two DOPE headgroups, as well as differences in lipid density presence or absence between the cryo-EM datasets. This is consistent with the published cryo-EM structure of SARS-CoV-2 Orf3a, in which the authors observed density in Lipid Site 1 and high-resolution density for the MSP1D1 scaffold protein, as we observe in our SARS-CoV-1 Orf3a map (Kern et al., 2021).

The possibility of two discrete sites for lipid binding may hint at a general function of Orf3a proteins that involves interaction with lipids in the bilayer. Intracellular vesicle formation is a cellular hallmark of a SARS-CoV-1 infection, possibly contributing to the region of viral RNA replication that is proposed to provide protection from host immune responses (Freundt et al., 2010). These intracellular vesicles are absent in Vero cells infected with SARS-CoV-1 ΔOrf3a, suggesting that Orf3a may be necessary for the formation of the vesicles (Freundt et al., 2010). Since the membrane vesicle structures are also observed during a SARS-CoV-2 infection, it is plausible that Orf3a may be required for this process and this has yet to be explored.

In addition to its interaction with lipids, we asked if there are host proteins that may bind to SARS-CoV-1 or SARS-CoV-2 Orf3a to better understand its possible contributions to viral pathogenesis. We turned to published proteomics datasets and focused on VPS39, a member of the HOPS membrane tethering complex that is essential for LE or AP fusion with Lyso (Gordon et al., 2020a; Balderhaar and Ungermann, 2013; Gordon et al., 2020b). A cellular defect common to HOPS complex mutants is an accumulation of LE and AP. We observe an enrichment of Rab7 puncta in cells overexpressing SARS-CoV-2 Orf3a_HALO, but not SARS-CoV-1 Orf3a_HALO. Consistent with this, VPS39 selectively interacts with SARS-CoV-2 Orf3a, and not SARS-CoV-1 Orf3a. By generating Orf3a chimeras (LC) we showed that an unstructured loop of SARS-CoV-2 Orf3a mediates binding to VPS39. Although SARS-CoV-1 Orf3a LC displays enhanced interaction with VPS39, it is not fully restored, and overexpression of the chimera is not sufficient to generate the Rab7 enrichment phenotype. Overall, our data suggests that the VPS39 interaction with Orf3a was acquired in SARS-CoV-2 and, by sequence and structural comparison between SARS-CoV-1 and SARS-CoV-2 Orf3a, that we have likely identified SARS-CoV-2 Orf3a’s novel region of interaction.

Concurrent with our cellular and biochemical studies connecting SARS-CoV-2 Orf3a overexpression with HOPS complex fusion defects, several other groups published comprehensive work that support our findings (Miao et al., 2021; Chen et al., 2021; Qu et al., 2021). In particular, Chen et al. surveyed the cytosolic domain of SARS-CoV-2 Orf3a to find a region of VPS39 interaction and identified a similar binding surface (Chen et al., 2021). Two SARS-CoV-2 Orf3a residues, S171 and W193, were identified to be important for mediating this interaction (Chen et al., 2021). Our Orf3a loop chimeras include the S171 substitution and their results support our conclusion that SARS-CoV-1 Orf3a LC can partially rescue the VPS39 interaction, but it is not sufficient to restore the SARS-CoV-2 Orf3a Rab7 phenotype (Chen et al., 2021). Chen et al. demonstrate that a W193 substitution into SARS-CoV-1 Orf3a can partly restore their SARS-CoV-2 Orf3a cellular phenotype, further delineating the SARS-CoV-2 Orf3a binding surface (Chen et al., 2021). A comparison of the surface electrostatic potential in this putative region of VPS39 binding highlights an acidic region of SARS-CoV-1 Orf3a which is neutral on the SARS-CoV-2 Orf3a surface (Figure 7B). We propose that the unstructured loop, W193 substitution, and the neutral charge in this region contribute to promoting the SARS-CoV-2 Orf3a and VPS39 interaction (Figure 7A-B).

How does the acquired interaction between SARS-CoV-2 Orf3a and VPS39 contribute to viral pathogenesis? Several recent studies have implicated the endocytic pathway as the primary mode of viral egress for several β-coronaviruses, including SARS-CoV-2, and that Orf3a may mediate this process (Ghosh et al., 2020; Chen et al., 2021). One possibility is that the SARS-CoV-2 Orf3a and VPS39 interaction promotes viral exit by perturbing forward endocytic trafficking through disrupting the HOPS complex-mediated fusion of LE with Lyso (Figure 7C). This could be due to (1) Orf3a sequestering VPS39 from the HOPS complex or, (2) Orf3a interacting with the HOPS complex through VPS39, an important distinction that has yet to be elucidated. A second, related possibility is that the SARS-CoV-2 Orf3a and VPS39 interaction promotes viral egress by interacting with known membrane tethering, fusion and trafficking complexes that facilitate lysosome movement to the cell periphery. In particular, VPS39 knockdown in SARS-CoV-2 Orf3a overexpressing cells reduces the localization of LAMP1 vesicles near the PM, and concomitantly diminishes recruitment of protein complexes involved in Lyso-PM trafficking (Chen et al., 2021). These data suggest that VPS39 is necessary for SARS-CoV-2 Orf3a-mediated Lyso-PM trafficking and implicates the recruitment of the HOPS complex by Orf3a in this process (Chen et al., 2021).

A third possibility is the SARS-CoV-2 Orf3a and VPS39 interaction prevents HOPS-mediated AP-Lyso fusion, which we and others have observed (Miller and Clapham, not shown, Figure 7C; Miao et al., 2021; Qu et al., 2021). Autophagy is an intracellular surveillance process that targets damaged cellular or foreign materials, such as viruses, for lysosomal degradation. Many viruses hijack host cell autophagy to prevent its degradation and promote survival. Overall, the acquired SARS-CoV-2 Orf3a and VPS39 interaction may function to assist with SARS-CoV-2 exit and host intracellular immune evasion. The molecular details of the host cell response to SARS-CoV-2 Orf3a need to be examined during viral infection to fully elucidate the contributions of Orf3a to SARS-CoV-2 cell physiology and pathogenesis.

All constructs and stable cell lines generated are available upon request. Atomic coordinates and cryo-EM density maps of have been deposited with the Protein Data Bank and Electron Microscopy Data Bank with the accession numbers: 8EQJ (SARS-CoV-2 Orf3a, LE/Lyso, MSP1D1 nanodisc; EMD-28538), 8EQT (SARS-CoV-2 Orf3a, PM, MSP1D1 nanodisc; EMD-28545), 8EQU (SARS-CoV-2 Orf3a, LE/Lyso, Saposin A nanodisc; EMD-28546) and 8EQS (SARS-CoV-1 Orf3a, LE/Lyso, MSP1D1 nanodisc; EMD-28544).

1. 1. Miller AN
   2. Houlihan PR
   3. Matamala E
   4. Cabezas-Bratesco D
   5. Lee GY
   6. Cristofori-Armstrong B
   7. Dilan TL
   8. Sanchez-Martinez S
   9. Matthies D
   10. Yan R
   11. Yu Z
   12. Ren D
   13. Brauchi SE
   14. Clapham DE

   (2023) RCSB Protein Data Bank

   ID 8EQJ. Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like membrane environment, MSP1D1 nanodisc.

   https://www.rcsb.org/structure/8EQJ
2. 1. Miller AN
   2. Houlihan PR
   3. Matamala E
   4. Cabezas-Bratesco D
   5. Lee GY
   6. Cristofori-Armstrong B
   7. Dilan TL
   8. Sanchez-Martinez S
   9. Matthies D
   10. Yan R
   11. Yu Z
   12. Ren D
   13. Brauchi SE
   14. Clapham DE

   (2023) Electron Microscopy Data Bank

   ID EMD-28538. Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like membrane environment, MSP1D1 nanodisc.

   https://www.ebi.ac.uk/emdb/EMD-28538
3. 1. Miller AN
   2. Houlihan PR
   3. Matamala E
   4. Cabezas-Bratesco D
   5. Lee GY
   6. Cristofori-Armstrong B
   7. Dilan TL
   8. Sanchez-Martinez S
   9. Matthies D
   10. Yan R
   11. Yu Z
   12. Ren D
   13. Brauchi SE
   14. Clapham DE

   (2023) RCSB Protein Data Bank

   ID 8EQT. Structure of SARS-CoV-2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc.

   https://www.rcsb.org/structure/8EQT
4. 1. Miller AN
   2. Houlihan PR
   3. Matamala E
   4. Cabezas-Bratesco D
   5. Lee GY
   6. Cristofori-Armstrong B
   7. Dilan TL
   8. Sanchez-Martinez S
   9. Matthies D
   10. Yan R
   11. Yu Z
   12. Ren D
   13. Brauchi SE
   14. Clapham DE

   (2023) Electron Microscopy Data Bank

   ID EMD-28545. Structure of SARS-CoV-2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc.

   https://www.ebi.ac.uk/emdb/EMD-28545
5. 1. Miller AN
   2. Houlihan PR
   3. Matamala E
   4. Cabezas-Bratesco D
   5. Lee GY
   6. Cristofori-Armstrong B
   7. Dilan TL
   8. Sanchez-Martinez S
   9. Matthies D
   10. Yan R
   11. Yu Z
   12. Ren D
   13. Brauchi SE
   14. Clapham DE

   (2023) RCSB Protein Data Bank

   ID 8EQU. Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc.

   https://www.rcsb.org/structure/8EQU
6. 1. Miller AN
   2. Houlihan PR
   3. Matamala E
   4. Cabezas-Bratesco D
   5. Lee GY
   6. Cristofori-Armstrong B
   7. Dilan TL
   8. Sanchez-Martinez S
   9. Matthies D
   10. Yan R
   11. Yu Z
   12. Ren D
   13. Brauchi SE
   14. Clapham DE

   (2023) Electron Microscopy Data Bank

   ID EMD-28546. Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc.

   https://www.ebi.ac.uk/emdb/EMD-28546
7. 1. Miller AN
   2. Houlihan PR
   3. Matamala E
   4. Cabezas-Bratesco D
   5. Lee GY
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   9. Matthies D
   10. Yan R
   11. Yu Z
   12. Ren D
   13. Brauchi SE
   14. Clapham DE

   (2023) RCSB Protein Data Bank

   ID 8EQS. Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc.

   https://www.rcsb.org/structure/8EQS
8. 1. Miller AN
   2. Houlihan PR
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   9. Matthies D
   10. Yan R
   11. Yu Z
   12. Ren D
   13. Brauchi SE
   14. Clapham DE

   (2023) Electron Microscopy Data Bank

   ID EMD-28544. Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc.

   https://www.ebi.ac.uk/emdb/EMD-28544

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Decision letter after peer review:

Thank you for submitting your article "The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kenton Swartz as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Youxing Jiang (Reviewer #1); Raimund Dutzler (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The three reviewers were enthusiastic and put forward a number of suggestions for improving the manuscript in revision. The following are the most important points to focus your efforts.

1) It would be valuable to provide a more detailed comparison of the determined structures with previous structures of SARS-CoV-2 ORF3a and to consider whether there are similarities to other proteins of known structure and function that might provide further insight into potential functional properties of ORF3a.

2) The quality of the presumed lipid density and its interaction with the protein is difficult to judge and its presentation should be improved. Although of potential relevance, the discussion of the lipid binding site seems exaggerated. The presence of lipids in a membrane protein is not unexpected and not every bound lipid is of functional importance. If you have additional evidence for the importance of the bound lipid, we encourage you to include that in the manuscript.

3) The interaction of MSP1D1 and Saposin A with the protein is somewhat unusual and we wondered whether there is anything that can be learned with respect to ORF3a function.

4) Please provide the information requested by reviewer #2 concerning reproducibility for some experiments and in one case a missing control.

Reviewer #2 (Recommendations for the authors):

1. Please include a control for Figure 1C showing a non-induced (non-Orf3a-expressing) cell.

2. Figure 1 – how many images/independent experiments are the images representative of? Please state in the legend.

3. Figure 2B – are these mean or exemplar traces? Please specify in the legend.

4. Figure 6 – how many independent experiments are the western blots/co-IPs representative of? Please specify in the legend.

Reviewer #3 (Recommendations for the authors):

The general quality of performed experiments is very high and I have only few remarks the authors should address.

• I think that a detailed comparison of the determined structures with previous structures of SARS-CoV-2 ORF3a should already be provided in the results. It would also be interesting whether the similarity to other proteins with a similar structure could provide any insight into potential functional properties.

• The quality of the presumed lipid density and its interaction with the protein is difficult to judge and its presentation should be improved.

• Although of potential relevance, I find the discussion of the lipid binding site exaggerated. The presence of lipids in a membrane protein is not unexpected and not every bound lipid is of functional importance. In case there is additional evidence, it should be provided.

• The interaction of MSP1D1 and Saposin A with the protein is unusual. I wonder whether there is anything that can be learned with respect to ORF3a function.

Essential revisions:

The three reviewers were enthusiastic and put forward a number of suggestions for improving the manuscript in revision. The following are the most important points to focus your efforts.

1) It would be valuable to provide a more detailed comparison of the determined structures with previous structures of SARS-CoV-2 ORF3a and to consider whether there are similarities to other proteins of known structure and function that might provide further insight into potential functional properties of ORF3a.

Our cryo-EM structures of SARS-CoV-2 Orf3a are nearly identical with the previously determined structures of SARS-CoV-2 Orf3a. In the discussion, we have clarified this by adding the global RMSD (0.38 Å) between our SARS-CoV-2 Orf3a PM MSP1D1 nanodisc and the previously published SARS-CoV-2 Orf3a structures in the text, and we also included a figure of our PM nanodisc model aligned with the 2.1 Å resolution published model (see Figure 3—figure supplement 3K).

In Kern et al., 2021, the authors also describe a tetrameric, low resolution (6.5 Å) cryo-EM structure of SARS-CoV-2 Orf3a.¹ We also observe a SARS-CoV-2 Orf3a protein population with a larger hydrodynamic radius by size-exclusion chromatography, similar to Kern et al. (pink trace, “4 mer”, Author response image 1).¹ However, we suspect that this oligomer may not represent a native assembly of the protein. We have yet to observe a SARS-CoV-2 Orf3a tetramer in isolated cell membranes by chemical crosslinking with either bismaleimidohexane (Figure 3—figure supplement 3I) or disuccinimidyl suberate (Miller and Clapham, unpublished). The cryo-EM structure of the SARS-CoV-2 Orf3a tetramer is formed by two Orf3a dimers. The point of interaction between the dimers is minimal, mediated by a cytosolic loop between β1-2. Among the residues within this loop is C153, which we suspect might be forming a non-specific disulfide bond during purification. In support of this, C153 is not present in SARS-CoV-1 Orf3a and, accordingly, we do not see a SARS-CoV-1 Orf3a protein population with a larger hydrodynamic radius by size-exclusion chromatography when purified in the same buffer and detergent conditions as SARS-CoV-2 Orf3a (green trace, “no 4 mer”, Author response image 1). Although more work would need to be done to convince ourselves on this matter, these lines of evidence begin to suggest that the SARS-CoV-2 Orf3a tetramer is formed by a non-specific disulfide bond during purification, and that it may not be physiologically relevant.

Author response image 1

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A distant relative of Orf3a proteins of coronaviruses is the M (membrane) protein, which is the most abundant protein in the virion and is critical for viral assembly and membrane budding.^,2,3 The M protein of SARS-CoV-1 was reported to assemble as a dimer and to adopt several conformations, a long and compact form, in the SARS-CoV-1 virion and in viral-like particles as visualized by low-resolution cryo-EM (>10 Å resolution) and cryo-ET.⁴ The M protein has also been shown to interact with the RNA-binding N (nucleocapsid) protein in SARS-CoV-1 and SARS-CoV-2.^5-8 Recently, three groups independently solved high-resolution X-ray and cryo-EM structures of dimeric M proteins from SARS-CoV-2 and a bat coronavirus, which have an overall fold that is shared with the Orf3a proteins.^9-11 In two of these publications, the authors also observe the long and compact conformations of the M protein, supporting the previous findings for the SARS-CoV-1 M protein.^{4, 10,11} Neither of these structures has a clear ion pore, and the M protein has not been reported to exhibit viroporin activity.^10,11 It is unclear what these conformations represent functionally. It was suggested from the SARS-CoV-1 M protein tomography studies that its long conformation may enforce rigidity and membrane curvature of the virion, which is ~100 nm diameter on average, and may facilitate binding to the N protein or direct interaction with viral RNA.⁴ The compact form is found in more elongated/flattened areas of the membrane, and its endodomain does not appear to contact the N protein and viral DNA.⁴

Superposition of SARS-CoV-2 Orf3a with the long and compact structures of the M protein yields an RMSD of 3.7 and 4.4 Å, respectively.¹⁰ Interestingly, the 50 Å diameter of the SARS-CoV-2 Orf3a homodimer is more in line with the diameter of M protein long conformation (50 Å) than the compact conformation (57 Å). We therefore wonder whether the totality of this evidence – the structural homology of Orf3a proteins with M protein and its similarity in diameter to the M protein long conformation, the proposed lipid binding sites that we observe in the Orf3a protein structures, and the reported function of SARS-CoV1 Orf3a to promote the formation of intracellular vesicles – could be consistent with a contribution to intracellular membrane remodeling.¹² Furthermore, Golgi fragmentation is another cellular hallmark of SARS-CoV-1 Orf3a overexpression, a phenotype which is shared with SARS-CoV-2 Orf3a based on our preliminary data (Matamala and Brauchi, unpublished) and a recent publication.^12,13 Overall, although the membrane remodeling hypothesis is intriguing, it is highly speculative and for this reason we have chosen to exclude this discussion from the manuscript.

2) The quality of the presumed lipid density and its interaction with the protein is difficult to judge and its presentation should be improved. Although of potential relevance, the discussion of the lipid binding site seems exaggerated. The presence of lipids in a membrane protein is not unexpected and not every bound lipid is of functional importance. If you have additional evidence for the importance of the bound lipid, we encourage you to include that in the manuscript.

Good point! We reinspected our Figures and we agree that the presentation of the lipid density needed to be improved. We have therefore modified Figure 4 to include DOPE molecules in Lipid Sites 1 (orange) and 2 (purple) in Figure 4A for visual clarity, and to be consistent with Figures 4B and C. In addition, we enhanced the contour level of the putative lipid electron density in Figure 4A-C to 7s from 2.5s and this significantly reduced the noise and improved visualization. In Figure 4D-I, we thickened the lines representing bonds for the DOPE molecules and side chains for clarity. In addition, after generating this figure for initial submission, we modified the Orf3a structures for PDB deposition by trimming the lipid acyl chains for more accurate representation. These updated models should have been used to generate the images in this Figure, and in this revision, we have therefore regenerated all the panels in Figure 4 with the PDB deposited models. These minor modifications do not change the interpretation of the data.

The functional significance of these lipids is unclear, and as the reviewer pointed out, the presence of lipids associated with membrane proteins is common. Although we believe that the lipids might have a physiological role and this could hint at a general mechanism of Orf3a protein function (see discussion of the M protein above), we agree that this may have been overinterpreted and we have therefore qualified some of the more speculative conclusions.

3) The interaction of MSP1D1 and Saposin A with the protein is somewhat unusual and we wondered whether there is anything that can be learned with respect to ORF3a function.

The membrane scaffold interaction appears to be shared between SARS-CoV-1 and SARS-CoV-2 Orf3a. Could this interaction be physiologically relevant and help us narrow our search for a common function of Orf3a proteins? We have wondered if this region of Orf3a could be an interaction hub for membrane tethering and/or integral membrane proteins. A known host membrane protein that was originally identified by mass spectrometry to interact with both SARS-CoV-1 and SARS-CoV-2 Orf3a proteins is CLCC1 (CLIC-like protein).^14,15 We have experimentally validated this by co-IP (Miller, Sanchez-Martinez, and Clapham, unpublished), and others have published a similar finding.¹⁶ CLCC1 is an ER-resident integral membrane protein that is annotated to be a chloride channel, CLIC-like 1, but as is the case with the channel function of Orf3a proteins, the experimental evidence for this is quite weak in our opinion.¹⁷ Furthermore, there is no atomic resolution structure of CLCC1. Although these data begin to hint at a general function for Orf3a proteins, we agree that the interaction hub hypothesis is highly speculative and the function of CLCC1 is unclear, so we chose to not include this discussion in the manuscript.

4) Please provide the information requested by reviewer #2 concerning reproducibility for some experiments and in one case a missing control.

We have addressed this by including a non-induced Orf3a_HALO control (Figure 1—figure supplement 1B) and have replaced Figure 1C with an image showing plasma membrane localization of Orf3a_HALO by TIRF (the original image was of Orf3a_SNAP). The Figure legends and text have also been updated to reflect these changes. All other points that were mentioned by reviewer #2 have been addressed and added to the Figure legends.

Citations:

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Author details

1. Alexandria N Miller

   Janelia Research Campus, Ashburn, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Patrick R Houlihan

   For correspondence

   millera@janelia.hhmi.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3986-7923

2. Patrick R Houlihan

   Janelia Research Campus, Ashburn, United States

   Contribution

   Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing

   Contributed equally with

   Alexandria N Miller

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2505-2347

3. Ella Matamala

   Physiology Institute and Millennium Nucleus of Ion Channel-Associated Diseases, Universidad Austral de Chile, Valdivia, Chile

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Deny Cabezas-Bratesco

   Physiology Institute and Millennium Nucleus of Ion Channel-Associated Diseases, Universidad Austral de Chile, Valdivia, Chile

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

5. Gi Young Lee

   Department of Biology, University of Pennsylvania, Philadelphia, United States

   Present address

   Department of Microbiology and Immunology, Cornell University, Ithaca, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

6. Ben Cristofori-Armstrong

   Janelia Research Campus, Ashburn, United States

   Present address

   Center for Advanced Imaging, University of Queensland, St. Lucia, Australia

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

7. Tanya L Dilan

   Janelia Research Campus, Ashburn, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3944-8385

8. Silvia Sanchez-Martinez

   Janelia Research Campus, Ashburn, United States

   Present address

   Molecular Biology Department, University of Wyoming, Laramie, United States

   Contribution

   Data curation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

9. Doreen Matthies

   Janelia Research Campus, Ashburn, United States

   Present address

   Unit on Structural Biology, Division of Basic and Translational Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States, United States

   Contribution

   Data curation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9221-4484

10. Rui Yan

    Janelia Research Campus, Ashburn, United States

    Contribution

    Data curation, Investigation, Writing – review and editing

    Competing interests

    No competing interests declared

11. Zhiheng Yu

    Janelia Research Campus, Ashburn, United States

    Contribution

    Supervision, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

12. Dejian Ren

    Department of Biology, University of Pennsylvania, Philadelphia, United States

    Contribution

    Supervision, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

13. Sebastian E Brauchi

    1. Janelia Research Campus, Ashburn, United States
    2. Physiology Institute and Millennium Nucleus of Ion Channel-Associated Diseases, Universidad Austral de Chile, Valdivia, Chile

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8494-9912

14. David E Clapham

    Janelia Research Campus, Ashburn, United States

    Contribution

    Supervision, Funding acquisition, Project administration, Writing – review and editing

    For correspondence

    claphamd@hhmi.org

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4459-9428

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