key: cord-0909806-xjd23pem
authors: Wong, Hoi Tong; Cheung, Victoria; Salamango, Daniel J.
title: Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism
date: 2021-12-07
journal: bioRxiv
DOI: 10.1101/2021.12.06.471415
sha: c5e5b521714f1ea4df1b241b967602fca77da570
doc_id: 909806
cord_uid: xjd23pem

Like many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds the nuclear pore components Nup98-Rae1 to inhibit nuclear translocation of activated STAT1/IRF3 transcription factors. However, a direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants can still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6/Nup98 interaction domain robustly inhibited nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral-membrane protein, opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication.

. Further investigation revealed that a direct interaction between the 67 C-terminal tail of ORF6 and the C-terminal domain of Nup98-Rae1 impairs docking of 68 cargo/receptor complexes to inhibit nuclear trafficking (Addetia et al., 2021; Kato et al., 69 2021; Miorin et al., 2020) . 70 While suppression of antiviral innate immune signaling is believed to be its 71 primary function, ORF6 also packages into nascent viral particles and induces 72 membrane structures resembling viral replication compartments (Huang et al., 2007; 73 Zhou et al., 2010) . These activities coincide with ORF6 subcellular distribution, as it 74 localizes to several membranous compartments during infection and when exogenously 75 expressed in several different cell types (Gunalan et al., 2011; Kumar et al., 2007) . Both 76 native and epitope tagged ORF6 proteins colocalize with markers for the Golgi 77 apparatus (Kato et al., 2021; Kopecky-Bromberg et al., 2007; Lei et al., 2020; Miorin et 78 al., 2020; Xia et al., 2020; Zhou et al., 2010) , endoplasmic reticulum (Kopecky-79 Bromberg et al., 2007; Lee et al., 2021; Lei et al., 2020; Zhou et al., 2010) , endosomes 80 (Gunalan et al., 2011; Kumar et al., 2007; Lee et al., 2021) , and nuclear envelope 81 (Addetia et al., 2021; Kato et al., 2021; Miorin et al., 2020) , which is consistent with the 82 current paradigm that ORF6 is a transmembrane protein capable of lateral diffusion 83 within membranous networks (Netland et al., 2007; O'Keefe et al., 2021; Zhou et al., 84 2010) . Taken together, these observations lend to an appealing model where ORF6 85 localization at the nuclear envelope facilitates a direct interaction with Nup98-Rae1 to 86 subsequently inhibit nuclear trafficking; however, this direct cause-and-effect 87 relationship has yet to be examined experimentally. 88 Here, we investigate the link between ORF6 localization and IFN antagonism. 89 Through an extensive panel of truncation and single amino acid substitution mutants in 90 combination with pharmacologic experiments, we demonstrate that ORF6 associates 91 with membranous compartments through two distinct structural determinants and is 92 most likely a peripheral-membrane protein. putative structural elements as a structure has yet to be solved (Fig. 1A) . Because the 113 C-terminal region of ORF6 is required for interacting with Nup98-Rae1 and for inducing 114 cytoplasmic accumulation of importinα (Frieman et al., 2007; Miorin et al., 2020) , we 115 reasoned that any cis-acting localization determinants reside upstream of this 116 protein/protein interaction domain. As expected, expression of amino acid residues 1-47 117 in HeLa cells resulted in localization comparable to wild-type, while expression of 118 residues 48-61 had no discernable localization pattern (Fig. 1B) . Unexpectedly, when 119 we further examined the 1-47 segment for cis-acting residues that dictate localization, 120 we were surprised to observe two distinct localization patterns. Expression of the first 121 half of this segment (amino acid residues 1-23) resulted in partial colocalization with the 122 Golgi marker, while expression of the second half (amino acid residues 24-47) resulted 123 in localization to an organelle distinct from the ER (Fig. 1B, 1-23 and 24-47, merge).

When the 1-23 segment was further truncated to residues 1-17, no localization pattern 125 was observed, suggesting that Golgi retention is partially mediated by amino acid 126 residues 18 IMRTFKV 24 (Fig. 1B) . 127 (Fig. 1B, 1 -47 and 18-47, merge) . These observations suggested that ORF6 maintains 135 steady-state localization through at least two distinct determinants, a longer protein 136 component from residues 1-47 that mediates steady-state membrane associations, and 137 a second region from 18 IMRTFKV 24 that dictates Golgi retention. To test this model, we 138 initially focused on 18 IMRTFKV 24 to determine if it harbors a Golgi retention motif.

Substitution of 18 IMRTFKV 24 to alanine in full length ORF6 not only disrupted Golgi 140 accumulation but induced freely diffuse intracellular puncta (Fig. 1C, 18-24Ala ). Further 141 investigation into the conservation of this motif revealed that SARS-CoV-1, bat, and 142 pangolin ORF6 proteins also require this motif to facilitate Golgi retention (Fig. 1C) . Of 143 note, SARS-CoV-1 and bat 18-24Ala mutants did not form intracellular puncta as 144 compared to SARS-CoV-2 and pangolin ORF6, but exhibited strong colocalization with 145 the ER marker, suggesting there is an inherent difference in the way these proteins 146 associate with membranes ( Fig. 1C , merge). It is possible this difference is attributable 147 to the putative helix from residues 24-47, as there is only ~50% amino acid identity 148 within this region across species (Fig. 1A) . 149 While no strict consensus motif has been defined for Golgi retention, numerous respectively. To test the contribution of these residues to Golgi retention, we generated 154 single and double amino acid substitution mutants at these positions and assessed 155 localization patterns. Independently exchanging these residues for glutamate had little 156 impact on ORF6 localization when expressed in HeLa cells; however, the RK20,23EE double mutant lost Golgi retention and localized to the ER-adjacent organelle, raising 158 the possibility that electrostatic interactions may facilitate ORF6 targeting to the Golgi 159 ( Fig. 1D , merge). This speculation is intriguing given that the Golgi membrane is 160 enriched with phosphatidylinositol-4-phosphate lipids, which unlike phosphatidylcholine, 161 ethanolamine, and serine, contain negatively charged headgroups.

Because several of the ORF6 mutants strongly localized to an ER-adjacent 163 organelle, we co-expressed relevant ORF6 mutants with markers for the most likely localization. To explore this possibility, we closely examined the amino acid composition 178 of these helices looking for clues as to how they might associate with membranous 179 compartments. We were surprised to discover that ORF6 exhibits a biased hydrophobic index and is predicted to be an amphipathic protein ( Figs. 2A and 2B ). From these 181 observations, we postulated two models to explain how ORF6 could be amphipathic 182 and localize to membranous compartments. First, ORF6 is a transmembrane protein 183 that forms higher order homo-or hetero-oligomers that shield the hydrophilic surface 184 from the hydrophobic membrane environment. Second, ORF6 is a peripheral-185 membrane protein that orients the hydrophilic helical surfaces toward the cytoplasm and 186 buries the hydrophobic portion within membrane surfaces.

To explore these models, we generated and tested single amino acid substitution 188 mutants that exchanged the wild-type residue for one with opposing biophysical (Fig. 3C) . Furthermore, the 236 Golgi marker only exhibited differential distribution in the presence of BFA, not digitonin, 237 indicating that the detergent was not grossly disrupting the structure of the Golgi. When

HeLa cells expressing SARS-CoV-2 ORF6 were treated with digitonin alone, no impact 239 on protein abundance or localization was observed (Fig. 3C) . However, when cells were 240 treated with BFA and then with digitonin, we observed significantly decreased 241 fluorescence intensity for ORF6 but not the Golgi marker (Fig. 3C) . Taken together, Nup98-Rae1 (Frieman et al., 2007; Kato et al., 2021; Kopecky-Bromberg et al., 2007; 251 Lei et al., 2020; Miorin et al., 2020; Xia et al., 2020) . The most likely scenario is that 252 ORF6 accumulation at the nuclear envelop facilitates a direct interaction with Nup98-253 Rae1 to block nuclear trafficking; however, this relationship has yet to be explored 254 experimentally. To determine if membrane association is required for inhibiting nuclear 255 trafficking, we examined a panel of localization disrupted mutants for their ability to 256 block eGFP-KPNA2 trafficking. To ensure that ORF6 function was not altered due to the 257 presence of the C-terminal mCherry tag, we generated a mCherry-T2A-ORF6 "self- 

Next, we wanted to confirm these mutants also block nuclear accumulation of activated 265 STAT1. To probe this activity, we treated A549 cells with type I IFN and assessed 266 STAT1 localization in the presence or absence of ORF6 proteins. As anticipated, all 267 mutants could inhibit nuclear accumulation of STAT1 at efficiencies comparable to wild-268 type (representative images in Fig. 4B, quantification in Fig. 4D) . Furthermore, we 269 assessed the ability of these mutants to inhibit interferon signaling using an IFNβ-eGFP for the Golgi marker. The next day, cells were treated as described above with the 362 following exception: digitonin treatment was not performed until after cells were 363 removed from culture plates to better preserve cell integrity. Following inhibitor treatment, cells were removed from plates using 0.025% Trypsin/EDTA solution, 365 centrifuged at 300 x g for 5 minutes, and then re-suspended in 2% FBS + 5 μM BFA/2 366 μM CHX in PBS. At this point, 50 μg of digitonin was added for 10 minutes and samples 367 were subjected to flow cytometry and analyzed using FloJo software. 

CoV-2 Accessory Proteins to Viral Pathogenicity in K18 Human ACE2 Transgenic Mice

Quantification of localization patterns for the indicated constructs in A549 cells either 485 stably expressing eGFP-KPNA2 or following IFN-treatment and STAT1 immuno-486 labeling. (E) Quantification of fluorescence from an IFNβ-eGFP reporter plasmid co-487 expressed with the indicated ORF6 proteins following RIG-I stimulation

Representative live cell fluorescence microscopy images of A549 cells expressing either 489 mCherry or the mCherry-3x-peptide construct. (G) Quantification of localization patterns

 Figure 4 . Membrane association of ORF6 is not required for interferon antagonism.(A and B) Representative live cell fluorescence microscopy images of the indicated mCherry-tagged proteins expressed in A549 cells stably expressing eGFP-KPNA2 (A) or following IFNtreatment and STAT1 immunolabeling (B). (C and D) Quantification of localization patterns for the indicated constructs in A549 cells either stably expressing eGFP-KPNA2 or following IFN-treatment and STAT1 immuno-labeling. (E) Quantification of fluorescence from an IFNb eGFP reporter plasmid coexpressed with the indicated ORF6 proteins following RIG-I stimulation.(F) Representative live cell fluorescence microscopy images of A549 cells expressing either mCherry or the mCherry-3x-peptide construct. (G) Quantification of localization patterns for the indicated constructs in A549 cells.