SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling

doi:10.21203/rs.3.rs-4486149/v1

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SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling

https://doi.org/10.21203/rs.3.rs-4486149/v1

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Open reading frame 3a (ORF3a) is among the most expressed viral accessory proteins of SARS-CoV-2, the pathogen responsible for the last pandemic. ORF3a mainly targets lysosomes of the host cell and promotes lysosomal inactivation through their deacidification, an essential step for lysosomal exocytosis and virus egress. However, the exact mechanism through which ORF3a performs this function is still unclear. While seminal studies suggested ORF3a functioning as a cation-selective viroporin, a recent work disproved this conclusion. To unravel the possible function of ORF3a, here we employed a multidisciplinary approach including molecular dynamics (MD), molecular biology, electrophysiology, and electron microscopy. Our electrophysiological results, in accordance with apreliminary MD structural analysis, ruled out that ORF3a functions as anion channel when expressed in HEK293 cells. Conversely, both MD and videoimaging experiments for the assessment of cell volume changes demonstrated that ORF3a mediates the transmembrane transport of water. Using MD, we also identified the putative selectivity filter for water permeation, and experimentally confirmed its relevance for water transport by showing that its mutation at the level of an asparagine (N82L and N82W) abolishes ORF3a-mediated water permeation. Finally, ORF3a expression in HEK293 cells determined lysosomal volume increase (swelling), mitochondrial damage, and accumulation of intracellular membranes, and these effects were reverted by the N82W mutation. Collectively, our data suggest a new function for the ORF3a protein as a lysosomal water permeable channel. The ORF3a-mediated water transport across lysosome membrane might promote lysosomal swelling and deacidification and, by consequence, inactivation, a key step to promote virus egress from the cell.

Biological sciences/Cell biology

Health sciences/Diseases

lysosomes

ORF3a

SARS-CoV-2

water transport

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a positive-strand RNA β-coronavirus that causes coronavirus disease-19 (COVID-19), responsible for the latest pandemic. Despite the numerous antiviral therapeutic strategies or vaccines under development, no fully effective anti-COVID-19 therapies have been developed to date. SARS-CoV-2 infection begins with the recognition and interaction of the S structural protein with the angiotensin-converting enzyme 2 (ACE2) expressed on the plasma membrane of the host cell. This interaction determines the subsequent entry of the virus into the cell. Once inside, the virus exploits the translation machinery of the host cell and intracellular organelles such as the endoplasmic reticulum (ER) and Golgi apparatus, to synthesize new viral particles and then escape from the cell^1,2. Given that the entry and egress processes are important steps in the viral infective cycle, the underlying molecular mechanisms may represent a potential target for the development of new vaccines or therapeutic strategies.

The SARS-CoV-2 genome expresses 29 proteins fundamental in the mechanism of virus replication, maturation, and egress, and classified as structural, non-structural, and accessories³. Two overlapping open reading frame (ORF) regions of the viral genome, ORF1a and ORF1b, encode for non-structural proteins (NSPs), which represent most of the proteins synthesized by the virus that mediate viral RNA replication. Additional genomic regions encode four structural proteins - spike (S), envelope (E), membrane (M), and nucleocapsid (N), that are components of the virion^4,5. In addition, the SARS-CoV-2 genome contains sequences encoding for a series of viral accessory proteins that participate in the entry and egress processes. Among these, ORF3a is one of the largest (274 amino acid residues) and the most expressed. It is prevalently localized in endosomes/lysosomes as an integral membrane protein, constituted by a luminal N-terminal domain, three transmembrane segments and a short cytoplasmic C-terminal domain⁶.

In recent years growing evidence has suggested that this protein plays a central role in virus egress from the host cell, by employing the late endosomes/lysosomes exocytic “non-conventional” pathway^7–10. Particularly, ORF3a abolishes the proteolytic/digestive power of lysosomes by promoting their swelling and deacidification and consequent inactivation^7,8, to be then employed as vesicles for virus egress through the lysosomal exocytosis process. In addition, ORF3a blocks autophagy by inhibiting the fusion of autophagosomes with lysosomes through its interaction with the VPS39 protein, a component of the homotypic fusion and protein sorting (HOPS) complex^9,11. The inhibition of the cell autophagic machinery prevents the digestion/destruction of viral particles.

Although these roles have been confirmed by several studies, the exact function of the ORF3a protein, and how it inactivates the proteolytic/digestive power of lysosomes through their deacidification, is still uncertain and elusive and a great effort is being made by many researchers to unveil its functional role. In 2021 two research groups suggested that ORF3a functions as a viroporin (i.e., viral ion channel) that mediates the passage of cations, such as Ca²⁺, Na⁺, and K^+ 6,12,13, a function that may possibly change the luminal content of lysosomes, causing their inactivation. However, a recent study has overturned this notion by demonstrating that ORF3a is not an ion channel¹⁴.

To unveil the possible function of ORF3a, in the present study we employed a multidisciplinary approach including molecular dynamics (MD), molecular biology, patch-clamp electrophysiology, and electron microscopy (EM). Collectively, the results we obtained showed that ORF3a is not an ion channel and suggested a new function of the protein as a lysosomal water permeable channel. The ORF3a-mediated water transport across lysosome membrane could induce lysosomal swelling and deacidification, a key step in promoting virus egress from the cell.

- HEK293 cell culture. HEK293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, high glucose, with L-glutamine, and Phenol red, Invitrogen, Life Technologies) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) and 1% Pen-Strep (100 U/mL, Sigma-Aldrich). The cell culture was maintained at 37°C with 5% CO₂.

- Plasmid constructions. The plasmid expressing ORF3a fused at its C-terminus to the EGFP was generated by amplifying the ORF3a coding region from cDNA of SARS-CoV-2-infected Vero cells using primers 1 and 2. ORF3a cDNA was digested with the restriction enzymes HindIII and BamHI and cloned into the vector pcDNA3-EGFP, upstream of the EGFP in the same restriction sites. The plasmid expressing the truncated form of ORF3a, lacking the first 41 amino acids, and fused to the N-terminus of the EGFP was obtained by amplifying ORF3a with primers 3 and 2 and cloned in the pcDNA3-EGFP vector as the previous plasmid.

Plasmids carrying the mutation of the asparagine 82 with either tryptophan or leucine were generated by overlap PCR using primers 5 and 6 or 7 and 8 for introducing tryptophan or leucine mutations, respectively. All constructs were sequenced to rule out the occurrence of nucleotide substitutions in the coding sequences.

- Transfection of HEK293 cells. HEK293 cells were plated in 6-well plates with cover slips with the aim of reaching 70-90 % confluency at the time of transfection, the day after (0.25 x 10⁶ cells for each well). The day of transfection the Lipofectamine^TM 3000 (Invitrogen, Life Technologies) was diluted in DMEM, supplemented with 10 % FBS and 1 % Pen-Strep, with the following condition: 7.5 µl of Lipofectamine^TM 3000 in 125 µl of medium. After, the master mix of DNA was prepared by diluting 5 µg of plasmid DNA, and 10 µl of P3000^TM reagent, in 125 µl of medium supplemented with 10% FBS, and 1% Pen-Strep. Subsequently, the master mix of DNA was added to the mix of Lipofectamine and mixed by gently pipetting several times. After an incubation time of 15 min, the DNA-lipid complex was added to the cells. HEK293 medium was changed after 6 h of incubation with the transfection master mix, and cells were incubated for 2-4 days at 37°C with 5% CO₂. Starting from the day after the transfection, it has been possible to detect the fluorescence signal of the plasmid transfected by using a fluorescence light microscope.

- Molecular Dynamics (MD) simulations. ORF3a protein models were based on the Cryo-EM structure, Protein Data Bank entry 6XDC. The initial atomic coordinates of the systems were obtained with CHARMM-GUI. The lipid membrane was a mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), with a ratio of 3-POPC/1-POPA. The channel was inserted in the lipid membrane as defined in the Orientations of Proteins in Membranes (OPM) database. The systems were solvated using TIP3P water molecules (∼15000 molecules) and ions to reach 200 mM KCl. VdW interactions were truncated at 9 Å. Standard scaling of 1−4 interactions was applied. The equilibration protocol consisted of 2000 energy minimization, followed by annealing of the system to 300 K temperature, then a 10 ns with a timestep equal to 2 fs. During the equilibration protocol, restraints were applied on the backbone of the protein. Long-range electrostatic interactions were calculated with the particle mesh Ewald method using a grid spacing of 1.0 Å. The SETTLE algorithm was used to restrain bonds with the hydrogen atom. The equilibration step was repeated by reinitializing the atoms velocities for additional 10 ns two times, having a final time of equilibration of 30 ns. The temperature was controlled at 300 K by coupling to a Langevin thermostat with a damping coefficient of 1 ps−1. A pressure of 1 atm was maintained by coupling the system to a Nose−Hoover Langevin piston, with a damping constant of 25 ps and a period of 50 ps. For molecular dynamics all simulations were performed without restraints with a timestep of 2 fs. NAMD2.11 and CHARM36 force field were used for all the simulations. Mutants N82W and N82L were generated with the plug-in “mutator” from VMD, then the resulting structure were minimized and equilibrated with the same protocol used for the WT protein.

- Immunofluorescence assay. For immunofluorescence assay, HEK293 monolayers were grown overnight, after transfection, on cover slips. Initially, they were washed twice with PBS, after they were fixed for 20 min with 4% (w/v) paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS for 20 min and blocked with 10% (v/v) FBS in PBS for 1 h. For primary antibody staining, mouse anti-GFP (monoclonal derived from hybridoma in culture; 1:200) were diluted in 2 % (v/v) FBS in PBS. Cover slips were washed three times for 5 min in 2% (v/v) FBS in PBS. Secondary antibodies used: goat anti-mouse Alexa Fluor 488 (Invitrogen, cat. # A11001). Secondary antibodies were used for 1 h diluted in 2% (v/v) FBS in PBS at 1:1000. Cover slips were then washed three times for 5 min in 2% (v/v) FBS in PBS and then mounted on slides with 25 ml of Vectashield® antifade mounting medium with DAPI (Vector Laboratories). Images were taken on a fluorescence light microscope (Nikon-Eclipse-TE2000-U) at 100x in oil immersion and analyzed using Fiji software (ImageJ).

- Electrophysiology. The whole cell dialyzed configuration was used for electrophysiological recordings. Currents were amplified with a HEKA EPC‐10 amplifier (List Medical), digitized with a 12‐bit A/D converter (TL‐1, DMA interface; Axon Instruments), and analysed with the software Patch‐Master package (version 2_60, ELEKTRONIK) and Microcal Origin 8.0. For on‐line data collection, macroscopic currents were filtered at 3 kHz and sampled at 200 μs/point. Patch electrodes of 3–5 MΩ were fabricated on PUL-100 Micropipette Puller (World Precision Instruments) from borosilicate glass (OD1.5 mm, ID 0.84 mm, WPI, Friedberg, Germany). Access resistances ranging between 5 and 15 MΩ were achieved by membrane disruption following seal formation and actively compensated to ~50%. For ion current recordings the internal solution contained: KCl 155 mM, EGTA-K 1 mM, MOPS 5 mM, MgCl₂ 1 mM, adjusted to pH 7.2 with KOH. The external standard Ringer's solution contained: NaCl 140 mM, KCl 5 mM, CaCl₂ 2 mM, MgCl₂ 2 mM, MOPS 5 mM, glucose 10 mM, adjusted to pH 7.4 with NaOH. For H⁺ current measurements both the internal and external solutions contained NMDG⁺ and MeSO^3- as the major cations and anions, respectively, similarly to what previously reported¹⁵. The internal solution contained: NMDG⁺ 100 mM, MeSO^3- 70 mM, MgCl₂ 2 mM, EGTA 10 mM, HEPES 100 mM, adjusted to pH 7.2 with 28 mM NMDG, with a theoretical calculated osmolarity of 314 mOsm. The external solutions contained: NMDG⁺ 100 mM, MeSO₃^- 70 mM, CaCl₂ 2 mM, MES 100 mM (pH 5.5) or 100 mM HEPES (pH 7.4). The osmolarity was adjusted to 314 mOsm by adding 14 mM NMDG-MeSO₃. All reagents were fresh daily solubilized at the concentrations stated, and bath applied with a gravity perfusion system. Experiments were carried out at room temperature (18-22°C).

- Cell volume measurements. Cells were perfused with an extracellular Ringer's solution identical to that used for electrophysiological experiments. For the study of the hypotonic‐induced cell swelling effect, a 30% hypotonic solution was applied. Relative projection area of cells was measured using a video‐imaging technique^16,17. The cells were observed through a 40× objective lens of a microscope connected to a video camera (Axiocam/Cm1, Zeiss). Images of the cells were saved as TIFF files and the area of each image was subsequently measured using ImageJ. Images were acquired every 10 s. Relative cell area (A_rel) was calculated by dividing the area of the cell at each time point (A_t) upon exposure to hypotonic solution by the initial area (A₀) taken under isotonic conditions (more precisely the average of the three time points recorded in the first 20 s).

- Fluorescence-Activated Cell Sorting (FACS). Cells were collected from cell culture plates with trypsin, resuspended in DMEM complete medium, and centrifuged at 900x g for 5 min. Afterward, cells were washed with PBS at 37°C and centrifuged as in the previous step. For sorting, cells were resuspended in Accumax - Cell Aggregate Dissociation Medium (Invitrogen, Thermo Fisher) and filtered with BD Filcon 70 μm (Fisher Scientific) to avoid clumping during the sorting process. GFP-positive cells were isolated using a FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA).

- Electron microscopy (EM). For EM preparation sorted transfected HEK293 cells were centrifuged at 10000x g for 5 min, washed 3 times with PBS, fixed with 3.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h and stored at 4°C. For embedding, cells were post-fixed in 2% osmium tetroxide (OsO₄) in the same buffer for 2 h and block-stained in saturated uranyl acetate replacement. After dehydration, specimens were embedded in epoxy resin (Epon 812). Ultrathin sections (about 40 nm) were cut using a Leica Ultracut R microtome (Leica Microsystem, Austria) with a Diatome knife (DiatomeLtd. CH-2501 Biel, Switzerland) and double-stained with uranyl acetate replacement and lead citrate. Sections were viewed and photographed in a 120 kV JEM-1400 Flash Transmission Electron Microscope (Jeol Ltd, Tokyo, Japan) equipped with CMOS camera Matataki and TEM Center software (Jeol Ltd, Tokyo, Japan).

- Quantitative analyses of EM images. Quantitative analyses of EM images. Quantitative analysis was done on random micrographs of HEK293 cell cultures. The average size of lysosomes was measured by drawing the outline of each lysosome using the Analy-SIS software of the EM digital camera (Olympus Soft Imaging Solutions). Only lysosome clearly membrane-limited were measured. The analyses were conducted on approximately 40 cells per sample.

- Statistical analysis. All statistical analyses were performed using the Origin v.8 software. To evaluate the significance of a difference, we used the two‐sample t‐test to compare two normally distributed populations. For multiple comparisons referred to a single control group, we used a one‐way analysis of variance test with post hoc Tukey's correction. For all experiments, p<0.05 was considered statistically significant and data are shown as mean ± SEM.

The structure and electrostatic properties of the ORF3a protein suggest that it is not an ion channel. In 2021, the SARS-CoV-2 ORF3a protein structure was reported by Kern and colleagues, who also suggested, in concomitance with another study by Toft-Bertelsen and others, that ORF3a functions as an unselective cation channel that allows the passage of Na⁺, K⁺, and Ca^2+6,13. This result was however confuted in the same year by Grant and Lester¹⁸ and two years later by the group of Clapham¹⁴, which showed that ORF3a does not form a cation permeable viroporin. Therefore, the exact function of this protein remains unclear. To unveil its possible function, we started by performing MD simulation on the ORF3a structure provided by Kern and co-workers to verify whether it has the right properties for a cation selective channel. ORF3a was inserted into a phospholipid bilayer, and the rest of the MD simulation box was filled with water, K and Cl ions (to give a final salt concentration of 150 mM).

The system was heated to 300 K and then equilibrated to 10 ns. In Fig.1A is reported, for the equilibrated structure, the backbone of the protein in cyan, while positively (lysine and arginine) and negatively (glutamate and aspartate) charged amino acidic residues are highlighted in yellow and red, respectively. The position of the positive charges, widely distributed at the intracellular entrance of the putative cation permeation pore (indicated), would make the passage of positive ions energetically unfavorable (Fig. 1A). This is confirmed by the analysis of the electrostatic potential profile of the ORF3a protein, shown along a plane perpendicular to the plasma membrane in Fig. 1B, clearly displaying the presence of a positive electrostatic potential at the entrance of the putative permeation pore. Consistent with the work of the Clapham’s group¹⁴, these preliminary structural observations suggest that the ORF3a protein is unlikely to function as a cation channel.

ORF3a is not an ion channel. We then tried to confirm the prediction of the MD structural analysis by performing electrophysiology to directly test the ability of ORF3a to behave as an ion channel. We first expressed, in HEK293 cells, the viral protein fused at the C-terminus with enhanced green fluorescent protein (EGFP) and looked at its expression and localization by immunofluorescence (Fig. 2A). Consistent with the ORF3a endoplasmic reticulum (ER)/Golgi/endolysosomal localization, the EGFP fluorescence signal displayed a large intracellular distribution (Fig. 2A, left).

Given our aim of measuring the ion currents of the plasma membrane through the patch-clamp technique, to enhance ORF3a localization on the cell surface, HEK293 cells were also transfected with an ORF3a deleted of the first N-terminal 41 amino acids, which we called truncated ORF3a protein (tORF3a). Consistent with previous studies^6,19, the removal of this sequence substantially increased ORF3a localization on the plasma membrane, as clearly shown by the enhanced fluorescence signal of cell surface exhibited by tORF3a-transfected cells (Fig. 2A, right), compared to ORF3a-transfected cells.

To evaluate whether the expression of the ORF3a protein correlated with the presence of an additional ion current on the plasma membrane, we performed electrophysiological experiments using the patch-clamp technique in the whole-cell dialyzed configuration. Ion currents were recorded at varying potentials following the application of different protocols, either voltage ramps of 1 second duration from -100 to 100 mV (Fig. 3B) or discrete steps of potential from -100 to 100 mV with incremental steps of 20 mV (Fig. 3C). As shown by both the representative current traces (Fig. 3B and C) and the quantitative analysis of the current-voltage (I-V) relationship of the steady state current (Fig. 3D), expression of the full-length ORF3a protein did not show any additional current compared to the control group (cEGFP). To demonstrate that the absence of a difference in current density was not the result of a low presence of the ORF3a protein on the plasma membrane, we repeated the same electrophysiological recordings on tORF3a cells. Again, the current density was not different from that recorded in control cells. The absence of ion currents when the 41 residues at the N-terminus were deleted, was consistent with a recent study showing that the same construct, which exhibited a robust cell surface expression, did not provide any additional currents compared to control cells¹⁹ Thus, together with the structural analysis performed on the ORF3a protein (Fig. 1), our electrophysiological results strongly suggest that ORF3a protein is not an ion channel.

ORF3a mediates water transport across the membrane. To obtain hints on a possible function of the ORF3a protein, we further analyzed MD simulations of the dimer placed in a physiological environment. Interestingly, we observed the presence of water columns through the transmembrane helices of each monomer, connecting the intra- and extracellular spaces (Fig. 3A and B). This was confirmed by the analysis of the distribution of water molecules within 10 Å of the protein, as a function of Z-axis perpendicular to the membrane, showing the presence of water throughout the transmembrane portion of the protein (Fig. 3B). To verify whether the ORF3a protein mediated a transmembrane water flux, we analyzed the MD simulation for the presence of water trajectories moving from one vestibule to the other one (Fig. 3C and D) and then counted the number of water molecules crossing the protein. The quantitative analysis revealed an average water conductance of ~2 molecules*ns^-1 (Fig. 3E), suggesting the presence of a water flux. Notably, all the water molecules permeating through the ORF3a protein occurred through one of the two water columns of the dimer, suggesting that differences might exist in the conformational properties of the two subunits. To experimentally demonstrate that ORF3a may function as a water permeable channel, we quantified the rate of water flux across the plasma membrane in our cell models. We designed an experiment in which the rate of cell volume increment (i.e., cell swelling) was used as a measure of water flux. To induce cell swelling, we perfused cells with an extracellular 30% hypotonic solution. Cell volume changes were monitored with a phase contrast microscope and images were acquired at 10 s intervals, for a total time of 6 min, a period sufficient to reach the peak of cell swelling. As shown by the average time course of cell volume changes, upon application of the hypotonic solution cells exhibited a first rapid and linear increase of cell volume, which lasted about 100-110 s from the beginning of the hypotonic shock, followed by a second less steep increment that reached a plateau (Fig. 3F). As described in Supplementary Information, the volume of a cell is expected to change linearly with time at the beginning of the cell swelling, with a slope proportional to the water permeability of the cell membrane. Therefore, the analysis of the slope of the linear part of the curve (red fitting line in Fig. 3F) would be an indication of the rate at which the water passes through the plasma membrane. We performed experiments of water permeability in control cEGFP cells and in cells expressing either the full-length (ORF3a) or the truncated (tORF3a) protein. The quantitative analysis of the relative volume taken during the first 100 s upon the exposure to the hypotonic solution revealed that the increase in cell volume strictly correlated to the ORF3a expression on the plasma membrane. Fitting the experimental data with a linear function, we observed that the slope of cell volume increase was 1.3 and 1.6 times greater in ORF3a and tORF3a than cEGFP cells, respectively (Fig. 3G).

Our experimental data confirmed the presence of a greater water flux in cells expressing ORF3a on the plasma membrane, confirming the prediction obtained by MD simulations and strongly indicating that ORF3a mediated the passage of water.

In principle, the presence of water columns connecting two compartments separated by a biological membrane would suggest that the ORF3a protein might also mediate the passage of protons. To verify this hypothesis, we designed an electrophysiological experiment aimed at recording proton currents. Specifically, we recorded currents in cEGFP and tORF3a cells, by using solutions devoid of the major permeable cations (i.e., Na⁺ and K⁺) and anions (Cl^-) (see Methods for additional details), at two different pH values (7.4 and 5.5). Results from these experiments clearly showed that changing the pH from 7.4 to 5.5 did not elicit any detectable current both in cEGFP and tORF3a cells, which showed a virtually identical basal conductance at two pH analyzed (Supplementary Fig. S1). This data indicated the absence of proton currents in cells expressing ORF3a on the plasma membrane, suggesting that ORF3a is not a proton channel.

Identification of the selectivity filter for the passage of water through ORF3a. The MD simulations reported in Fig. 3A, indicated the presence of a preferential route for the passage of water through the protein, represented by the water columns between the helices connecting the intra- and extracellular vestibules. Here, we used the HOLE program²⁰ to identify the permeation pore and its surrounding residues. In Fig. 4A is reported the ORF3a dimer where the two monomers of the protein are indicated as monomer 1 and monomer 2, and the permeation pores for both monomers is shown with different colors indicating a hole radius above 2.8 Å (blue, there was room for two or more water molecules), of 1.4-2.8 Å (green, there was room for a single water), and lower than 1.4 Å (red, the pore radius was too narrow for a water molecule). The identified holes showed that only monomer 1 allowed the proper passage of water from one vestibule to the other, as clearly visible by the absence of red regions alongside the permeation pore. Conversely, monomer 2 showed restriction zones that prevented the correct transit of water molecules. The different water permeability between the two monomers depended on the spatial disposition of two specific residues represented in orange, the phenylalanine at position 120 (F120) and leucine at position 83 (L83) (Fig. 4A). In monomer 1, the arrangement of the two residues facilitated the passage of water. In fact, at this level the permeation pore calculated with the HOLE program was much larger than the diameter of a water molecule.

On the other hand, in monomer 2, as better visible in the 180° rotated protein, the spatial positioning of the two residues created a bottleneck (the hole radius was labelled red) that hindered the passage of water. In the water permeable pore, immediately under the F120 and L83 residues, it was possible to observe a transition area between the intra- and extracellular environments, in which the permeation pore in both monomers was marked in green, according to the HOLE program, characterized by the presence of specific aminoacidic residues marked in red, that entered in direct contact with all permeating waters. These four residues included two asparagines at positions 82 and 119 (N82 and N119), one phenylalanine at position 79 (F79), and an isoleucine at position 123 (I123) (Fig. 4A, bottom). Given the localization of these residues in the transition zone between the intra- and extracellular sides and since at this level the permeation pore coincided with the dimensions of a single water molecule, we hypothesized that these four amino acids could constitute a possible selectivity filter for water. We have also identified the amino acid residues that entered in contact (at less than 3 Å distance) with the permeating water molecules during their passage through the permeation pore. As shown in the bar plot of Fig. 4C, the highest number of detections was exhibited by both N82 and N119 residues. This data strongly indicated that these two residues might be essential for the constitution of the putative selectivity filter for water. To confirm this notion, we performed MD simulations of a mutated ORF3a dimer where the asparagine at 82 was substituted with either leucine or tryptophan, to create the N82L and N82W mutant proteins (Fig. 4D). Results from our MD simulations showed that both mutations disrupted the ability of the ORF3a to transport water (Fig. 4E). These results were confirmed by water permeability experiments (i.e., cell volume changes measurements) performed in cells transfected with the wild-type truncated ORF3a (tORF3a-WT) or with truncated ORF3a carrying both mutations (tORF3a-N82W and ORF3a-N82L). Consistent with the MD predictions, these experiments demonstrated that substituting the asparagine at position 82 either with leucine or tryptophan completely abolished the ability of the ORF3a protein to mediate the passage of water, since the slope during hypotonic-induced cell swelling was approximately half in cells expressing both the mutated tORF3a proteins (N82L and N82W) compared to that of cells expressing the WT protein (Fig. 4F), a difference very close to that observed between cEGFP and tORF3a cells (Fig. 3G).

ORF3a-WT transfected cells exhibit enlarged lysosomes. ORF3a localizes at lysosomes, where it induces their increase in diameter and deacidification with the consequent inactivation of the proteolytic enzymes^8,21. Lysosomal inactivation, together with the inhibition of autophagic pathways, is the prerequisite for virus egress via the lysosomal-mediated exocytosis^7–9,22. Since we had strong indication that ORF3a can mediate the passage of water, and since lysosomal swelling can be the reason for an increased lysosomal diameter and can lead to a strong reduction or suppression of lysosomal lytic power²¹, we investigated possible ultrastructural alterations/modifications of lysosomes caused by the presence of the ORF3a protein and related to its function as a water transporter. To this aim, we first analyzed lysosome morphology through ultrastructural EM analyses in control cEGFP cells and in cells transfected with the full-length ORF3a-WT protein. In electron micrographs, lysosomes are usually identified as spherical organelles appearing as single membrane-limited rounded vesicles of about 0.05 to 0.5 µm in diameter, which may present a granular, electron-dense content due to the presence of digested material. Autolysosomes are instead double-membrane limited organelles of variable shape derived from the fusion of lysosomes with autophagosomes/amphisomes when digestion starts^23,24. Interestingly, as shown in Fig. 5, differently from cEGFP cells where lysosomes had the standard described morphology, that is circular vesicles usually containing spots of electron dense granular material (Fig. 5A and inset), in cells transfected with the full-length ORF3a-WT, lysosomes often appeared as enlarged white/clear circular vesicles, lacking electron-dense material inside (Fig. 5B and inset). The EM quantitative analysis fully supported the visual observation. Indeed, we quantified the average lysosomes size (area, µm²) and found that ORF3a-WT cells exhibited lysosomes with an average size (0.28±0.06 µm²) almost three times greater than that of lysosomes of in cEGFP-transfected cells (0.10±0.01 µm²) (Fig. 5D). We also evaluated one of the two mutant ORF3a proteins, the ORF3a-N82W and found that lysosomes were abundantly present and had the standard morphology (Fig. 5C). Interestingly, in ORF3a-N82W cells, the size of lysosomes was even smaller than in control cEGFP cells (0.06±0.008 µm²).

In the process of examining lysosomes, we also observed additional intracellular alterations in ORF3a-transfected cells including large double-membrane structures with materials inside and a striking remodeling of intracellular membranes (likely ER/Golgi) to form a labyrinth of a quite regular pattern resembling a honey-comb structure. In addition, mitochondria appeared structurally abnormal, with an irregular bean-shaped morphology, and with an aberrant internal matrix. Notably all these structural alterations were never found either in control cEGFP nor in ORF3a-N82W cells (Supplementary Fig. S2).

The present study examines the possible functional role of the SARS-CoV-2 ORF3a accessory protein. First, by analyzing the structure of the dimer, it appears clear that ORF3a has neither the typical architecture of ion channels expressed by eukaryotic cells nor the electrostatic properties to allow the permeation of cations. In accordance, electrophysiological experiments do not show any additional basal current in cells expressing ORF3a compared to control cells, as also found by Clapham’s group¹⁴. Unexpectedly, though, MD simulations demonstrate the formation of water columns between the transmembrane helices of each monomer of the protein, which connect the intra- and extracellular environments and generate a net flux of water molecules across the membrane at a rate of ~ 2 molecules*ns^− 1, close to that found for mammalian aquaporins²⁵. The transit of water molecules occurs through a specific permeation pore and their passage from one vestibule to the other one is mediated by four specific amino acid residues that form a putative selectivity filter. Notably, mutation of residue N82, belonging to this putative selectivity filter, disrupts the transit of water, excluding the possibility that the increase in water permeability in ORF3a-expressing cells is due to the modulation of water-permeable channels endogenously expressed in the host cell and strongly favoring the idea that the ORF3a protein itself functions as a water transporter. A limitation of this conclusion is that both electrophysiological and video-imaging experiments have been done in cells expressing the protein on the plasma membrane. It is widely recognized that ORF3a mainly localizes to the endomembrane system, mainly to the ER and endosomes/lysosomes, where it plays its pathophysiological roles during virus life cycle. Although ORF3a has also been observed on the plasma membrane, it is possible that this localization is only a consequence of lysosomal exocytosis and that ORF3a does not play any functional role in this cellular compartment. Therefore, both H⁺ and water transport measurements performed directly on lysosomes will offer important information for establishing the function of the ORF3a protein. However, a strong indication that ORF3a may function as water transporter in lysosomes is given by our EM results showing that ORF3a expression resulted in lysosomal volume increase (swelling), an effect reverted by mutations that inhibit water transport. Collectively, these data identify a new function of the ORF3a protein as a water-permeable channel, which can possibly be relevant for SARS-CoV-2 viral cycle.

ORF3a is not an ion channel, but a water transporter. To date, the exact function of the ORF3a accessory protein is still unclear and under debate. In recent years, divergent claims have been reported on the ability of the ORF3a protein to function as a viroporin (weak or non-selective ion channels expressed by several viruses, which play a key role in viral life cycle). Seminal studies postulated ORF3a from both SARS-CoV-1 and SARS-CoV-2 being a highly hydrophobic, virus-encoded protein forming a nonselective cation channel with a preferential permeability for Ca²⁺ ions^6,12,13. The initial studies by Kern et al.⁶ and Toft-Bertelsen et al.¹³ showed that ORF3a forms a viroporin in Xenopus oocytes and liposome membranes, although Grant and Lester, in the same experimental preparation, were unable to see any ORF3a-dependent conductance when the transcript was expressed in the plasma membrane¹⁸. A more comprehensive study was recently published by the Clapham's group, which unequivocally demonstrates that ORF3a is not an ion channel¹⁴. Using three high-resolution cryo-EM structures of the ORF3a of SARS-CoV-2 under different conditions, these authors observed a constriction within the transmembrane region of the protein and the presence of a positively charged aqueous vestibule, which would strongly discourage cation permeation. Furthermore, they were unable to record ORF3a-dependent ion currents in several mammalian cells, as well as in other experimental preparations, including Xenopus oocytes, in reconstituted systems using purified ORF3a, and in endo/lysosomes from HEK293 cells. Clapham and colleagues downgraded the ionic currents through ORF3a reported by Kern et al. and by Toft-Bertelsen et al. to technical artefact, such as an unusually high protein to lipid ratio in reconstituted systems⁶ or contamination with endogenous channels¹³.

In line with the results of Clapham and colleagues, we also found that ORF3a does not function as an ion channel. This conclusion is based on the following lines of evidence. First, analysis of the structure of ORF3a protein shows that the putative ion permeation pore is too narrow to accommodate a dehydrated cation. Second, the position of positively charged amino acid residues, widely distributed at the intracellular entrance of the putative cation permeation pore, would make cations passage thermodynamically extremely difficult. Third, electrophysiological data show no ionic current from the ORF3a-enriched plasma membrane of HEK293 cells (obtained using the N-terminal deletion construct that promotes protein expression in the plasma membrane)¹⁴. Finally, MD simulations do not show passage of cations through the ORF3a protein.

Instead, we observed a novel formation consisting of continuous water columns between the transmembrane helices of the ORF3a protein monomers, which connect the intra- and extracellular environments. This finding stems from the analysis of the distribution of water molecules along the protein axis perpendicular to the membrane plane, showing uninterrupted water molecules throughout the transmembrane portion of the protein. MD simulations also showed trajectories of water moving from one vestibule to the other (at a rate of ~ 2 molecules per nanosecond). These MD results are confirmed by video-imaging experiments aimed at assessing the passage of water across the plasma membrane of HEK293 cells transfected with ORF3a. Namely, slope analysis of the initial increase in cell volume following the application of a hypotonic stress shows that the rate of volume change increases in parallel with the tendency of the transfected ORF3a construct (cEGFP vs ORF3a vs tORF3a) to localize to the cell membrane.

Regarding the passage of water through the protein, we identified a preferential route and a putative selectivity filter formed by four amino acid residues located in the transition zone between the intra- and extracellular environments. Of these, two asparagines, at positions 82 and 119 (N82 and N119), are essential for the formation of the putative water selectivity filter, in that they show the highest rate of interaction with water molecules crossing the transition zone between the two vestibules, and mutations of one of them – asparagine 82 – with tryptophan (N82W) or leucine (N82L) disrupt the ORF3a protein's ability to transport water, as assessed by both MD simulation analyses and video-imaging experiments.

Interestingly, MD shows that only one of the two subunits of the dimer constituting ORF3a displays a pore having the right dimensions to allow water movement from one to the other vestibule, and indeed only in one of the two subunits an effective permeation of water molecules can be observed. This because of the presence of two hydrophobic residues, F120 and L83, whose three-dimensional conformation differs among the two subunits, the two residues being much closer in the non-conducting subunits, so to create a narrow constriction hampering the passage of water molecules. Notably, in longer preliminary MD simulations (data not shown) we could observe rapid changes in the F120/L83 lateral group distance, suggesting that it might represent a form of rapid gating. Further studies of this process are guaranteed.

Possible physio-pathological implications of the ORF3a-mediated water transport. Generally, the virus lifecycle comprises different major stages, ranging from virus attachment/entry into the host cell to its release. As far as the SARS-CoV-2 is concerned, while the early stages of the infectious cycle have been relatively well defined, the mechanisms underlying late stages (i.e., virus egress) are instead less well understood. A recent paper by Ghosh and colleagues, reveals that the egress process of β-coronaviruses, including SARS-CoV-2, is based on a lysosomal, Arl8b-dependent exocytosis pathway and requires lysosomal inactivation through a deacidification (alkalinization) process ⁷. In SARS-CoV-2 the lysosomal alkalinization, which is essential for their inactivation and consequent fusion with the plasma membrane, is known to be mediated by ORF3a^8,9,21. However, the mechanism(s) through which ORF3a promotes lysosome deacidification and exocytosis is still debated and poorly understood.

Our electrophysiological experiments discard the possibility that ORF3a directly mediates H⁺ extrusion, given the complete absence of recordable H⁺ currents in HEK293 cells expressing ORF3a on the plasma membrane. The question thus arises of how a water-permeable channel can dissipate a H⁺ gradient and deacidify a lysosome. Lysosomes are intracellular organelles whose basal water permeability is generally low, as suggested by a study showing that the introduction of synthetic water channels to lysosomal membrane dramatically increases osmotic tension of lysosomes²⁶, and by the finding that a VRAC-mediated water flux, promoted by hypotonicity, leads to lysosome swelling^27,28. Our EM data suggests that the ORF3a expression may promote an osmotic flux of water into the hyperosmotic lysosomal lumen, causing lysosomal swelling. In smooth muscle cells, Busch and colleagues reported that hypotonic-induced lysosome swelling induces lysosome alkalinization²⁹. Although not experimentally proven, the authors supported the idea that the rapid increase in lysosomal pH, following exposure to osmotic shock, would result from increased H⁺ leak across lysosomal membrane or from lysosomal fusion with other organelles that have a higher pH than the lysosomes themselves²⁹. This notion would support our hypothesis that the increased ORF3a-dependent lysosomal water permeability would lead to lysosomal swelling and deacidification.

Another possible role of the ORF3a water transport in the SARS-Cov-2 lifecycle could be related to the exocytosis-mediated virus egress. Indeed, it has been reported that lysosomes express Ca²⁺ permeable TRPML2 channels that can be activated in response to lysosome swelling³⁰. Thus, it is possible that a TRPML2-dependent Ca²⁺ release from lysosomes participates in their fusion with the plasma membrane (i.e. lysosomal exocytosis) in ORF3a-expressing cells, and that ORF3a-mediated water transport is needed for triggering the Ca²⁺-dependent lysosome exocytosis. The idea that lysosomes increase their fusion with the plasma membrane during osmotic shock-induced lysosomal swelling has been widely reported^27,28.

Conclusion. The results obtained in this study confirm that ORF3a is not a cation channel (a viroporin). They also show for the first time that ORF3a harbors uninterrupted water columns between its transmembrane helices, making it function as a water-permeable channel. This new feature could provide insight into the mechanism by which ORF3a promotes deacidification and inactivation of lysosomes, a necessary step for virus egress through the lysosomal exocytotic pathway. A better knowledge of the molecular mechanisms by which the ORF3a protein promotes viral egress may hopefully lead to new antiviral therapies.

The results obtained in this study confirm that ORF3a is not a cation channel (a viroporin). They also show for the first time that ORF3a harbors uninterrupted water columns between its transmembrane helices, making it function as a water-permeable channel. This new feature could provide insight into the mechanism by which ORF3a promotes deacidification and inactivation of lysosomes, a necessary step for virus egress through the lysosomal exocytotic pathway. A better knowledge of the molecular mechanisms by which the ORF3a protein promotes viral egress may hopefully lead to new antiviral therapies.

Author contributions: A.M. and L.C. conceived and designed the study and wrote the manuscript. A.M. and L.S. performed all the electrophysiological and video-imaging experiments, analyzed the results, and prepared the figures. R.F., M.V.L., and A.DiB. performed cell culture preparations and transfections and obtained immunofluorescence images; G.R. prepared samples for EM analysis; S.V. sorted EGFP positive cells used for EM preparations and edited the manuscript; S.B. performed EM experiments, analyzed results, prepared figures, and edited the manuscript; M.DiC. designed all the plasmids used in the study, supervised all molecular biology preparations, and edited the manuscript.

Competing interests. The authors declare no competing interests.

Acknowledgments. This study was supported by two grants to Luigi Catacuzzeno: Fondazione Cassa di Risparmio di Perugia (Project #J95F21000050007) and MUR PRIN 2022 (grant 20223XZ5ER, CUP J53D23006940006).

Data availability. All relevant information supporting the findings of this study is presented in the manuscript and supplementary materials.

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SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling

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