bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Endomembrane systems are reorganized by ORF3a and Membrane (M) of SARS-CoV-2

2 3 Yun-Bin Lee1, Minkyo Jung2, Jeesoo Kim3, Myeong-Gyun Kang1, Chulhwan Kwak1,5, Jong-Seo Kim3,4,*, Ji- 4 Young Mun2,*, Hyun-Woo Rhee1,4,*

5

6 1Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea 7 2Neural Circuit Research Group, Korea Brain Research Institute, 41062 Daegu, Republic of Korea 8 3Center for RNA research, Institute for Basic Science, Seoul 08826, Republic of Korea 9 4School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea 10 5Department of Chemistry, Ulsan National Institute of Science and Technology, 44919 Ulsan, Korea 11 12 *Corresponding authors: 13 14 Dr. Hyun-Woo Rhee 15 Email: [email protected], 16 17 Dr. Ji-Young Mun 18 Email: [email protected]. 19 20 Dr. Jong-Seo Kim 21 Email: [email protected], 22

1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 23 Summary

24 The endomembrane reticulum (ER) is largely reorganized by severe acute respiratory syndrome coronavirus

25 2 (SARS-CoV-2). SARS-CoV-2 ORF3a and membrane (M) protein expression affects ER-derived structures

26 including cubic membrane and double membrane vesicles in coronavirus-infected cells; however, the

27 molecular mechanisms underlying ER remodeling remain unclear. We introduced a “plug and playable”

28 proximity labeling tool (TurboID-GBP) for interactome mapping of GFP-tagged SARS-CoV-2 ORF3a and M

29 proteins. Through mass spectrometric identification of the biotinylated lysine residue (K+226 Da) on the viral

30 proteins using Spot-TurboID workflow, 117 and 191 proteins were robustly determined as ORF3a and M

31 interactomes, respectively, and many, including RNF5 (E3 ubiquitin ligase), overlap with the mitochondrial-

32 associated membrane (MAM) proteome. RNF5 expression was correlated to ORF3a ubiquitination. MAM

33 formation and secreted proteome profiles were largely affected by ORF3a expression. Thus, SARS-CoV-2

34 may utilize MAM as a viral assembly site, suggesting novel anti-viral treatment strategies for blocking viral

35 replication in host cells.

36

37 Highlights

38 • SARS-CoV-2 proteins ORF3a and M alter endoplasmic reticulum proteome profile

39 • ORF3a affects mitochondrial-associated membrane formation

40 • SARS-CoV-2 may utilize mitochondrial-associated membrane as viral assembly site

41 • ORF3a and M interactome proteins may serve as targets for COVID-19 treatment

42

43 eTOC Blurb

44 ER remodelling by SARS-CoV-2 ORF3a and M protein

45

2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 46 INTRODUCTION 47 The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory

48 syndrome coronavirus 2 (SARS-CoV-2) necessitates detailed research into the molecular mechanisms

49 underlying the ability of this virus to infect and colonize host cells. In particular, understanding the mechanism

50 by which each viral protein encoded by the SARS-CoV-2 genome interacts with host factors is essential for

51 the prevention or treatment of COVID-19. Similar to SARS-CoV and Middle East respiratory syndrome

52 coronavirus (MERS), SARS-CoV-2 has been classified in the genus Betacoronavirus of the Coronaviridae

53 family (Qin et al., 2005). It encodes 16 non-structural proteins (NSP1–16), 4 structural proteins (spike protein

54 [S], membrane protein [M], envelope protein [E], and nucleocapsid protein [N]), and at least 9 accessory

55 proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10). The non-structural

56 proteins are generated from an auto-proteolytic process and participate in the formation of the replicase-

57 transcriptase complex, which is essential for the viral life cycle (Gordon et al., 2020). The structural proteins

58 are components of each viral particle and are key factors in host cell infection (Gordon et al., 2020). The

59 accessory proteins have functions associated with interferon and immune responses; for example, ORF3a of

60 SARS-CoV activates the NOD-like receptor protein 3 (NLRP3) inflammasome (Siu et al., 2019), and ORF3b

61 of SARS-CoV-2 acts as an interferon antagonist (Konno et al., 2020).

62 A previous study has demonstrated viral protein–host protein interactions of SARS-CoV-2 using a co-

63 immunoprecipitation (Co-IP) method followed by mass spectrometry analysis (Gordon et al., 2020). The Co-

64 IP can show interaction partners with strong binding affinity to the viral proteins of interest (vPOI); however,

65 these interactome data may also include artificial protein-protein interactions that do not occur in cells since

66 these experiments are conducted using cell lysates isolated in artificial lysis buffer conditions. To overcome

67 these limitations, proximity labeling (PL) methods have recently been developed, which are based on an

68 enzymatic biotinylating reaction using in situ-generated reactive biotin species by genetically expressed

69 enzymes. Examples of these enzymes include engineered ascorbate peroxidase (APEX) (Rhee et al., 2013),

70 promiscuous biotin ligase (BioID) (Roux et al., 2012), and TurboID, which is an engineered biotin ligase with

71 faster kinetics (labeling time less than 30 min) (Branon et al., 2018). Since the biotinylation reaction induced

72 by the PL enzymes occurs in living cells, and the labeling radius of the reactive biotin species (biotin-phenoxy

3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 73 radical and biotin-AMP) is less than 50 nm, mass spectrometry analyses of endogenous proteins biotinylated

74 by the PL enzymes can accurately reflect the physiological interactome of the POI that is genetically expressed

75 alongside these enzymes.

76 PL is being used to identify the host interactome of viral proteins, including coronaviruses (V'Kovski et al.,

77 2019) and SARS-CoV-2 (Laurent et al., 2020; Samavarchi-Tehrani et al., 2020; St-Germain et al., 2020).

78 Although this method shows various important host proteins that support viral life cycles (such as eIF3 and

79 eIF4 complexes) and anti-viral proteins (such as MAVS, PKR, and LSM14A/B), the results from this

80 technique may contain false positive findings due to the conventional mass detection workflow. Currently,

81 most PL studies (including the viral interactome studies) have used conventional mass detection of non-

82 biotinylated peptides of the streptavidin-bead (SA-bead) enriched proteins from the PL-labeled cell lysate.

83 However, this workflow can misidentify artificial interacting proteins in the lysis buffer as biotinylated

84 proteins (false positive) when those proteins are reproducibly shown in the biotinylated samples over the

85 controls. Furthermore, recent reports describing the mapping of the SARS-CoV-2 interactome (Laurent et al.,

86 2020; St-Germain et al., 2020) utilized BioID, which performs slow biotinylating reactions on proximal

87 proteins over a 12-h period. This long biotinylation reaction may perturb the physiological conditions of host

88 cells.

89 To maximize the advantages of recently improved PL methods, in this study, we combined TurboID, a fast

90 biotinylating enzyme (Branon et al., 2018), with the Spot-BioID method (Lee et al., 2019). In this technique,

91 cell lysates are digested with trypsin followed by SA-bead enrichment at the peptide level, so that only peptides

92 modified by TurboID with biotin-conjugated lysine (K+226) are detected using mass spectrometry. We further

93 assessed the ability of our modified PL method, dubbed Spot-TurboID, to analyze proteins biotinylated by

94 TurboID, and to ultimately obtain in vivo host interactome information without false positive results.

95 In the present study, we aimed to identify which proteins located near the endoplasmic reticulum (ER)

96 interact with SARS-CoV-2 proteins, as the ER membrane is proposed to be the primary replication site for

97 SARS-CoV such as SARS-CoV-2 (Stertz et al., 2007). Folding and synthesis of membrane, lipids, secretory

98 proteins, sterols, and ion storage occurs in the ER (Lin et al., 2008). Among SARS-CoV-2 proteins, ORF3a is

99 associated with vesicle sorting and fusion mechanisms while the M protein interacts with membrane-shaping

4 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 100 proteins (St-Germain et al., 2020). Therefore, we focused on the ORF3a and M proteins, using our modified

101 PL system to examine their functions in living cells. We conducted correlative light and electron microscopy

102 (CLEM) imaging experiments to determine the localization and targeting sites of these proteins in living cells.

103 These findings can offer new research directions to improve our understanding of the mechanisms underlying

104 SARS-CoV-2 infection and to identify new therapeutic targets for COVID-19.

105

106 Results

107

108 GFP-tagged SARS-CoV-2 structural and accessory proteins target specific organelles

109 To confirm the subcellular localization of the SARS-CoV-2 proteins, we prepared 11 constructs of vPOIs

110 (NSP7, NSP8, NSP9, M, N, ORF3a, ORF3b, ORF6, ORF7b, ORF9b, and ORF9c) tagged with GFP using a

111 13-amino acid linker (GAPGSAGSAAGSG) (Figure 1A). Confocal imaging of HEK293-AD cells showed

112 that the GFP-tagged vPOIs were well expressed (Figure 1B). Compared with non-structural proteins,

113 structural and accessory proteins targeted specific organelles. NSP7, NSP8, and NSP9 showed a whole-cell

114 expression pattern, indicating that these proteins were diffused throughout the cell, and do not specifically

115 interact with a certain organelle, possibly due to the lack of viral RNAs. In contrast, M, N, ORF3a, ORF3b,

116 ORF6, ORF7b, ORF9b, and ORF9c showed organelle-specific localization patterns, indicating that these

117 proteins interact with host cell proteins of specific organelles. Among these constructs, we observed that the

118 expression patterns of ORF3a and M merged well with an ER marker protein, SEC61B (Figure S1A).

119 In line with a previous study showing that N protein is associated with stress granules (Gordon et al., 2020),

120 confocal imaging of HEK293-AD cells co-expressing N-GFP and G3BP1-V5 (stress granule marker protein)

121 (Anderson and Kedersha, 2006; Nover et al., 1983), showed that the two patterns clearly merged, representing

122 a stress granule pattern (Figure 1C). In addition, we confirmed that GFP-tagged vPOIs (i.e. ORF3a, ORF6,

123 ORF7, M) were merged with Flag-tagged vPOIs in co-expressed cells, indicating that tagging with GFP (~27

124 kDa) is acceptable for those vPOIs like as tagged with small epitope tag (Flag) (Figure S1B). These

125 experiments confirmed that the GFP-tagged structural and accessory protein constructs of SARS-CoV-2

126 established were suitable for our further interactome study.

5 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 127

128 The GFP/GBP binding system is applicable for identifying the SARS-CoV-2 interactome in live cells

129 As the POI-GFP and APEX2-GFP binding protein (GBP) system was previously validated for GFP-specific

130 electron microscope (EM) imaging (Ariotti et al., 2015), we utilized this GFP and GBP binding “plug and

131 play” system for the interactome analysis of vPOI-GFP by co-expression of TurboID-GBP via Spot-TurboID

132 workflow (Figure 2A). To confirm a specific binding between vPOI-GFP and TurboID-GBP, M, N, ORF3a,

133 ORF6, and ORF7b were selected. Confocal images of HEK293-AD cells co-expressing vPOI-GFP and

134 TurboID-GBP were identical to those of cells expressing vPOI-GFP alone (Figure 2B, S2), demonstrating

135 that GFP and GBP bound well, and the patterns of the biotinylated proteins merged with the GFP and GBP

136 patterns. Thus, we could confirm that co-expressed TurboID-GBP has a strong and specific binding interaction

137 with GFP-tagged vPOIs and it did not appear to interrupt the targeting of vPOI-GFP to specific organelles.

138 In the western blot analysis of proteins biotinylated by TurboID-GBP and detected using streptavidin-

139 horseradish peroxidase (SA-HRP), we observed that the biotinylated protein patterns of TurboID-GBP were

140 altered when it was co-expressed with GFP-tagged M, N, ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF9b,

141 ORF9c, or ORF10 proteins (Figure 2C, D, S3A-C). The change in SA-HRP western blot patterns of the

142 biotinylated proteins by the PL enzyme (Lee et al., 2016) may reflect that the neighboring proteins around

143 TurboID-GBP were significantly altered when TurboID-GBP was translocated to the co-expressed vPOI-GFP

144 in the same cell. These experiments also showed that the GFP-tagged structural and accessory proteins of

145 SARS-CoV-2 may be surrounded by different host proteins in cells, and that these proteins may be readily

146 biotinylated by TurboID and identified using mass spectrometry via the Spot-TurboID workflow (Figure 2A).

147

148 ORF3a and M SARS-CoV-2 proteins destructively perturb the ER membrane organization.

149 Among the structural and accessory proteins that exhibit an ER targeting pattern, ORF3a and M contain 3

150 transmembrane (TM) domains (Figure 3A, B, S4A-B) and both proteins showed a similar alteration of protein

151 band patterns in western blot analysis, with a bigger molecular weight than expected (Figure 2D). ORF3a

152 shows 85.1% sequence similarity with ORF3a of SARS-CoV (Gordon et al., 2020), which exhibits ion

153 transport activity and induces NLRP3 inflammasome activation (Siu et al., 2019). ORF3a has also been shown

6 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 154 to possess pro-apoptotic activity (Ren et al., 2020). Similarly, M of SARS-CoV-2 shows 96.4% sequence

155 similarity with M of SARS-CoV (Gordon et al., 2020), which was reported to target the ER, ER-Golgi

156 intermediate compartment, and Golgi apparatus, and is associated with apoptosis (Chan et al., 2007) and

157 nuclear factor-(NF) κB signaling (Fang et al., 2007). We hypothesized that these proteins may perturb the

158 ultrastructure of endomembrane systems, as it has been shown that many viral proteins target the

159 endomembrane systems of host cells (Inoue and Tsai, 2013).

160 To test this hypothesis, we prepared ORF3a-APEX2 and M-APEX2 constructs, with APEX2 tagged to the

161 C-terminus of the vPOIs (Figure 3A), and used them for EM imaging in HeLa cells following APEX-mediated

162 diaminobenzidine (DAB) staining (Martell et al., 2012). ORF3a and M proteins largely disrupted the ER

163 (Figure 3C, D; Figure S5A-B). ORF3a-APEX2 expression significantly increased cubic membrane (CM,

164 also denoted as convoluted membrane) structure formation; moreover, these CM structures were generated in

165 ORF3a-GFP expressing cells shown by CLEM imaging (Figure 3C, S5C). Notably, CM structures also form

166 in coronavirus-infected cells (Deng and Angelova, 2021; Snijder et al., 2020), and they are considered as neo-

167 organelles for the assembly of viral proteins (Uchil and Satchidanandam, 2003). Additionally, in ORF3a-GFP

168 expressed HeLa cells, structure of double membrane vesicle (DMV, red arrows in Figure 3C) was discovered

169 near ER and mitochondria. Formation of DMV by ORF3a-APEX2 was reproduced in A549 lung cell line

170 (Figure 3E, G). We confirmed that these CM and DMV structures were not observed in SEC61B-APEX2 or

171 APEX2 transfected controls (Figure S6A-C). Since DMV formation is believed to house replication

172 complexes of various RNA viruses (Cortese et al., 2020; Klein et al., 2020; Wolff et al., 2020), our result

173 indicates that ORF3a of SARS-CoV-2 plays an important role in the formation of ER-derived neo-organelles

174 (i.e. CM and DMV) that are essential for viral replication in host cells.

175 Similarly, ER was clearly disrupted in M-expressing cells (Figure 3D) and appeared to curl into whorl

176 patterns (also termed as aggresomes). These patterns were also observed in herpes simplex virus-infected cells

177 (Nii et al., 1968) and ER stress-induced cells (Schuck et al., 2014; Snapp et al., 2003). In CLEM imaging of

178 M-GFP (Figure S5D), we observed the formation of multilamellar bodies (MLB) at the ER membrane (Figure

179 3D, F) and electron-dense autophagic vesicles (green arrows in Figure S5D). The biogenesis of MLB

180 regulated is by autophagy (Hariri et al., 2000) and MLBs are utilized during extracellular vesicle-type viral

7 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 181 egress (Bunyavirus) (Sanz-Sánchez and Risco, 2013). In flavivirus replication (such as Zika virus), autophagy

182 is usually activated and autophagic vesicles accumulate inside the cells in in cellulo (Hamel et al., 2015) and

183 in vivo (Cao et al., 2017) experiments. In our study, these structures were not detected in non-transfected HeLa

184 (Figure S5B) and A549 cell controls (Figure S6A). Moreover, we confirmed that the structure of ER, Golgi,

185 and mitochondria did not change in SEC61B-APEX2 or APEX2-expressed cells (Figure S6B-C). Our EM

186 results therefore imply that the ORF3a and M proteins of SARS-CoV-2 may largely contribute to the

187 generation of ER-derived neo-organelles (Fernández de Castro et al., 2014; Tenorio et al., 2018; Xie et al.,

188 2019) for efficient viral assembly and replication by remodeling the endomembrane systems of host cells. To

189 identify the host proteins involved in this process, we conducted an in cellulo interactome analysis of the

190 SARS-CoV-2 ORF3a and M proteins using GFP-tagged constructs with TurboID-GBP via the Spot-TurboID

191 workflow.

192

193 ORF3a and M interactomes showed perturbed proteomic landscape at the ER membrane.

194 For mass spectrometric analysis, we used the aforementioned Spot-TurboID method (Figure 2A) to obtain

195 an accurate interactome map of the ORF3a and M proteins. To obtain the biotinylated peptidome, HEK293T

196 cells co-expressing vPOI-GFP and TurboID-GBP and control HEK293T cells co-expressing GFP and

197 TurboID-GBP (Figure 4A) were treated with biotin (50 μM, 30 min) in full 10% fetal bovine serum medium,

198 washed with cold Dulbecco’s phosphate-buffered saline, and lysed using 2% sodium dodecyl sulfate in 1×

199 Tris-buffered saline. Lysate samples were trypsin-digested prior to SA-bead enrichment for biotinylated

200 peptidome enrichment. Biotinylated peptides were isolated from the SA-beads and injected to the Orbitrap

201 Fusion Lumos mass spectrometer. A total of 4232, 3818, and 6299 peptide-spectrum matches containing

202 biotin-moiety peptides at the lysine residue (K+226) were detected in the ORF3a-GFP + TurboID-GBP, M-

203 GFP + TurboID-GBP, and unconjugated GFP (control) + TurboID-GBP expressing cells, respectively.

204 Based on the reproducibility and similarity values of the biotinylated peptidome of each sample (Lee et al.,

205 2016), replicate samples under the same conditions were clustered together, confirming the reproducibility of

206 the experiment (similarity value > 0.84, Figure S7A-D). From the volcano plot analysis of mass signal

207 intensity of proteins biotinylated by TurboID-GBP, proteins that were more strongly biotinylated in the

8 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 208 ORF3a-GFP and M-GFP co-expressed samples versus the control GFP-expressed sample were selected as

209 interactome proteins of ORF3a and M, respectively (log2(fold change, FC) ≥ 2.3 and –log10 p-value ≥ 2.3,

210 Figure 4B, S7E). The major population of the interactomes for ORF3a and M comprises endomembrane

211 system proteins, suggesting that SARS-CoV-2 ORF3a and M are primarily localized at the ER.

212 Among the 117 filtered proteins for the ORF3a interactome, 40.2% were located at the plasma membrane

213 and 37.6% were located at the endomembrane (Figure 4C, S7F). Among the 191 filtered proteins of the M

214 interactome, 49.7% were located at the plasma membrane and 25.1% were located at the endomembrane

215 (Figure 4C, S7F). Thus, a large proportion of plasma membrane and endomembrane proteins were present in

216 both, the ORF3a and M interactomes. According to gene ontology analysis, proteins associated with regulation

217 of localization and ubiquitin-dependent ER-associated protein degradation (ERAD) pathway were highly

218 enriched among the 117 proteins of the ORF3a interactome (Figure 4D, S7G). Proteins related to localization

219 and nitrogen compound transport were highly enriched among the 191 proteins comprising the M interactome

220 (Figure 4D, S7G). From these analyses, we could see that the molecular functions of ORF3a and M are highly

221 related to localization and transport of other substances within the endomembrane of host cells.

222 To further our understanding of these interactions, we then filtered the top 30 most strongly biotinylated

223 proteins (red or blue-colored dots in Figure 4B) among the ORF3a and M interactomes, respectively. The 30

224 filtered proteins of the ORF3a interactome were classified according to localization and function (Figure 4E).

225 Surprisingly, 13 of these 30 proteins (43.3%) overlapped with the recently identified mitochondrial-associated

226 membrane (MAM) proteome determined using Contact-ID (Kwak et al., 2020). This suggests that ORF3a of

227 SARS-CoV-2 is localized at the MAM, in line with a previous host interactome analysis of SARS-CoV-2

228 using the Co-IP method (Davies et al., 2020). Since the MAM-enriched ORF3a interactome proteins are

229 associated with vesicle transport (BET1, BET1L, SAR1A, and VAMP3), regulation of membrane integrity

230 (EPHA2, JAM3, and SEC63), and lipid synthesis and steroid binding/processes (DHCR7 and SCD5), we

231 postulated that ORF3a may be involved in the regulation of vesicle trafficking at the MAM to support viral

232 assembly and egress processes similar to other viral proteins that target the MAM, such as the NS5A and

233 NS5B proteins in hepatitis C virus (Hamamoto et al., 2005). Moreover, several ion transport proteins that are

234 part of the endomembrane system (such as CLCC1, SCD, SLC16A1, SLC22A5, TMEM110, and TMEM165)

9 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 235 were identified in the ORF3a interactome. Based on previous findings that ORF3a of SARS-CoV regulates

236 the ion channels at the ER membrane (Chan et al., 2009; Issa et al., 2020) and ORF3a of SARS-CoV-2 forms

237 Ca2+ and K+ channels (Kern et al., 2020), we postulate that ORF3a may localize at the sub-domain of ER

238 where ion transport proteins cluster.

239 Similarly, the top 30 filtered proteins of the M interactome were classified by localization and molecular

240 function (Figure 4F). Among these proteins, we found the proteins associated with cell cycle/apoptosis

241 (ITGB1, IRS4, WDR6, and STEAP3) to be highly enriched in our M interactome. We also observed that

242 several vesicle transport proteins (BET1L, SAR1A, VAMP3, and YKT6) were highly enriched in the M

243 interactome, which may influence ER vacuolization and apoptosis associated with the M protein (Chan et al.,

244 2007) (Figure 3D, F).

245 Moreover, several known viral interacting host proteins, such as CXADR and ZC3HAV1, were found in

246 the M interactome. Interestingly, infection of SARS-CoV-2 induced an increase in the transcript level of

247 CXADR (coxsackievirus and adenovirus receptor) (Dörner et al., 2004; Martino et al., 2000) in a SARS-CoV-

248 2 infected patient sample (Zhou et al., 2020). Among our M interactome proteins, ZC3HAV1 (CCCH-type

249 zinc-finger antiviral protein) inhibits viral replication by recruiting other proteins responsible for sensing

250 numerous viral RNAs (Kerns et al., 2008; Oshiumi et al., 2015), including human immunodeficiency virus-

251 (HIV)-1 (Zhu et al., 2011), Moloney and murine leukemia virus (Lee et al., 2013), and xenotropic MuLV-

252 related virus (Wang et al., 2012b). Notably, ZC3HAV1 was also enriched in the ORF3a interactome. Thus, our

253 data suggest that ZC3HAV1 is also involved in the anti-viral response against SARS-CoV-2.

254

255 Spot-TurboID reveals topology of TM proteins interacting with ORF3a and M

256 Since ORF3a and M are targeted to ER membrane (Figure S1A), it is reasonable that majority of their

257 interacting proteins are membrane residents such as endomembrane proteins or plasma membrane proteins,

258 synthesized at the ER membrane: ORF3a: 78%, M: 75% (Figure 4C). For membrane proteins, identification

259 of their membrane topologies is crucial to understanding their domain-wise function at either side of the

260 membrane. In our work, membrane topological information of the biotin-labeled membrane proteins was

261 easily extracted. Using our Spot-TurboID method, we obtained mass spectrometry data that included the

10 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 262 biotinylation sites of each digested peptides. All digested peptides isolated following SA-bead enrichment had

263 at least one biotin-modified site at a lysine (K) residue. Since the biotinylation by TurboID-GBP occurred via

264 cytosolic regions of the protein, biotinylated sites on proximal interacting proteins of vPOI-GFP logically

265 faced the cytosol. We then obtained membrane topological information for proteins harboring a TM domain

266 using mass spectrometry data. It is noteworthy that using similar approaches, we successfully revealed the

267 topologies of inner mitochondrial membrane (Lee et al., 2016) and MAM proteins (Kwak et al., 2020).

268 Using this workflow, 22 TM proteins were identified in each of the ORF3a and M interactomes and the

269 biotinylation sites and topology of these proteins are shown in the figures (Figure 5, S8A-B). For example,

270 the biotinylation sites of SEC61B and VAMP3 (K20 of SEC61B, K35 of VAMP3) from the ORF3a

271 interactome are well matched to previously characterized topologies. Based on these results confirming the

272 reliability of the Spot-TurboID data, we propose the hitherto unknown topologies of multiple ORF3a and M

273 interacting membrane proteins (CLCC1, DHCR7, TMEM110, FKBP8, RNF5, UBR3, and UBIAD1) (yellow-

274 colored proteins in Figure 5).

275 It is noteworthy that this detailed topological information can be only obtained by direct detection of

276 biotinylated peptides, however, conventional methods that overlook these biotin-PTM signatures cannot report

277 it. We believe that our data provide detailed information on the domains heading toward cytosolic domain of

278 ORF3a and M proteins at the membrane, which lays foundation for further studies on host membrane proteins.

279 Notably, among the biotinylated membrane proteins, several lysosome-associated proteins (i.e., VPS16,

280 VMA21, ITM2C, TMEM165, and RAB7A) were identified in the ORF3a interactome. This finding is

281 consistent with recent reports that ORF3a hijacks the HOPS complex and RAB7, which are necessary for

282 membrane contact between autophagosomes and lysosomes for autolysosome formation (Miao et al., 2021;

283 Zhang et al., 2021). Our result implies that ORF3a may dynamically localize from the MAM to the

284 autophagosome and autolysosome because autophagosome formation is also known to be initiated at the

285 MAM (Hamasaki et al., 2013).

286

287 RNF5 ubiquitinylate ORF3a of SARS-CoV-2

288 In regard to the ORF3a interaction with membrane proteins at the autophagosome and lysosome, RNF5, which

11 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 289 is strongly biotinylated by Spot-TurboID (Figure 6A), interests us because it is an ER-anchored E3 ubiquitin-

290 protein ligase involved in regulating the autophagic process during bacterial infections (Kuang et al., 2012).

291 Interestingly, RNF5 is involved in the inflammatory response (Li et al., 2019), and it regulates the antiviral

292 response by degrading an outer mitochondrial protein, MAVS (Zhong et al., 2009; Zhong et al., 2010), which

293 implies that RNF5 might function at the MAM. Indeed, RNF5 was identified as an MAM-resident E3 ubiquitin

294 ligase in our recent study (Kwak et al., 2020).

295 Since the molecular function of RNF5 with SARS-CoV-2 is not fully characterized yet, we conducted

296 further studies to reveal its functional relationship with SARS-CoV-2 proteins. From confocal microscopy

297 experiments, we observed that cytoplasmic dispersed Flag-conjugated RNF5 was largely altered by ORF3a

298 and M expression and co-localized with the respective viral proteins (Figure 6B-C, S9A). This result indicates

299 that RNF5 might be activated by ORF3a and M protein expression. From western blot analysis after Co-IP,

300 we observed that overexpression of RNF5 induced further ubiquitination of ORF3a protein (Figure 6D, S9D-

301 E). It is noteworthy that overexpression of a mutant RNF5(C42S) whose ubiquitin-transferring ring finger

302 domain is not functional owing to C42S mutation (Zhong et al., 2009) did not induce ubiquitination of ORF3a

303 (Figure S10). These results indicate that RNF5 is involved in the ubiquitination of viral proteins and their

304 interacting host proteins (Figure 6E).

305 In our study, results of western blot analysis revealed increased expression of ORF3a and M following

306 MG132 treatment (Figure S9B-C). Thus, we propose that RNF5 is responsible for ubiquitination and

307 degradation of ORF3a and M proteins derived from SARS-CoV-2. Our result is in a good agreement with a

308 recent study that selected RNF5 as a specific ubiquitin ligase for viral proteins of SARS-CoV-2 from genome-

309 wide screening (Zhen Yuan, 2021). Together with our result, we expect RNF5 to be an effective target for the

310 development of anti-viral therapeutics for SARS-CoV-2.

311

312 SARS-CoV-2 ORF3a influences ER–mitochondria contact sites and the secreted proteins of host cells

313 Interestingly, we also observed that many of the ORF3a and M interactome proteins overlapped with our

314 recently identified MAM proteins. We have identified 115 local resident proteins using Contact-ID, which

315 utilized the reconstituted biotinylating activity of split BioID fragments at the MAM, which is an ER and

12 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 316 mitochondrial contact site (Kwak et al., 2020). Twenty-eight of the proteins that filtered out from the ORF3a

317 interactome (23.9%) and the 24 from the M interactome (12.6%), overlapped with the 115 proteins previously

318 identified to be a part of the MAM proteome. A total of 18 proteins overlapped between ORF3a, M, and the

319 MAM proteome (Figure 7A). The complete list of proteins in the ORF3a and M proteomes that overlap with

320 the MAM proteome, classified by function, are given in Figures 7B and 7C, respectively. Since many of the

321 ORF3a interactome proteins overlapped with those from the MAM proteome rather than the M interactome,

322 we questioned whether ORF3a could more strongly hinder MAM formation.

323 To address this, we used the Contact-ID tool to monitor changes in the proteomic composition at the MAM

324 in response to ORF3a expression (Figure 7D). (Contact-ID generated more biotinylated proteins in the

325 ORF3a-expressing sample compared to that in the control sample (blue arrows in Figure 7E; Figure S11A-

326 D), which implies that ORF3a may lead to a considerable change of the proteomic landscape at the MAM. We

327 also found that ORF3a was biotinylated by Contact-ID (red arrows in Figure 7E). Thus, we are currently

328 conducting further investigations to analyze which proteins are recruited to the MAM in response to ORF3a

329 expression. Similar to ORF3a, we observed that M was also biotinylated by Contact-ID (Figure S12).

330 We speculate that the expression of vPOI may change the secretory proteome because our EM results

331 showed that ORF3a and M largely perturb the endomembrane system and many viral infections have been

332 known to affect secretory proteome profiles (Lietzén et al., 2019). To investigate alterations in the host cell

333 secretome, we used recently developed method dubbed iSLET (in situ secretory protein labeling via ER-

334 anchored TurboID) (Kim et al., 2020). In the iSLET method, SEC61B-TurboID is utilized to biotinylate

335 secreted proteins at the ER luminal side near to the SEC61 translocon channel since most secreted proteins

336 transit through the ER lumen via the SEC61B translocon channel. Secretory proteins biotinylated in SEC61B-

337 TurboID-expressing cells can be detected in the culture media or mouse bloodstream. In this study, we utilized

338 the iSLET method to assess whether the secretome profiles of the host cells changed in response to ORF3a

339 and M expression (Figure 7F).

340 In the SA-HRP western blot results, the amounts of biotinylated proteins in the culture media of cells co-

341 expressing SEC61B-TurboID and ORF3a were substantially higher compared to that of the control sample

342 lacking ORF3a expression (Figure 7G, S13A-B). In addition, we found ORF3a in the culture media of the

13 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 343 cells (red arrows in Figure 7H, S13C). In whole cell lysates, ORF3a was also biotinylated by SEC61B-

344 TurboID (Figure S14). Similar to ORF3a, we observed that the amounts of biotinylated proteins increased in

345 the secretome of M-expressed cells (Figure S15A), and M protein was also secreted from cells (Figure S15B)

346 and was biotinylated by SEC61B-TurboID (Figure S15C).

347 These results imply that ORF3a and M may upregulate cargo secretion from the ER and there may be a

348 specific sorting pathway for the secretion of ORF3a in the ER lumen, as has been shown for other secreted

349 viral proteins (Alefantis et al., 2005). In the ORF3a interactome, SAR1A/B and USE1 are known to play

350 essential roles in cargo secretion at the ER-Golgi interface (Petrosyan et al., 2015) and further investigation is

351 required to identify whether the interaction of these vesicle transport proteins with ORF3a could induce ER-

352 derived vesicle formation and changes in the secretome profile.

353

354 Discussion

355 In this study, we demonstrated that ORF3a and M perturb the integrity of endomembrane systems in host

356 cells, thereby resulting in devastating effects. As shown in other viruses (Dengue, porcine epidemic diarrhea,

357 and Zika viruses) that utilize the ER for replication (Lee et al., 2018; Monel et al., 2017; Wang et al., 2012a;

358 Zou et al., 2019), including SARS-CoV (Fung et al., 2014), our data show that ORF3a and M of SARS-CoV-

359 2 largely affect membrane integrity and the proteomic landscape at the ER. Although we employed ectopic

360 expression method of ORF3a and M protein constructs to ensure biosafety, a recent study shows that ectopic

361 expression of ORF3a of SARS-CoV-2 is enough to show pathogenesis of SARS-CoV-2 in Drosophila (Shuo

362 Yang, 2020). Since some of the proteins we identified, including RNF5, are also highlighted as viral host

363 proteins in other studies of SARS-CoV-2 (Zhen Yuan, 2021), we believe that our interactome data reflects

364 physiological networks of the ORF3a and M proteins of SARS-CoV-2.

365 During our interactome analyses of ORF3a and M, we observed a notable overlap with the MAM proteome

366 (Kwak et al., 2020). Among the overlaps, VAP-A and VAP-B interact with NS5A and NS5B to promote viral

367 replication and protein assembly of hepatitis C virus at the MAM (Hamamoto et al., 2005). We confirmed that

368 RNF5 (Kuang et al., 2012), co-localized with ORF3a and induced ubiquitination of ORF3a (Figure 6). This

369 result is consistent with previous studies that showed ubiquitination of SARS-CoV-2 ORF3a (Cao et al., 2021)

14 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 370 and RNF5-induced ubiquitination of SARS-CoV-2 M (Zhen Yuan, 2021). Whether overexpression or chemical

371 activation of RNF5 could be used as a novel anti-viral or anti-inflammatory strategy for SARS-CoV-2 warrants

372 further investigations.

373 In our study, RNF5 was identified as part of the ORF3a interactome, whereas previously reported

374 interactome mapping approaches such as conventional proximity labeling technique (Samavarchi-Tehrani et

375 al., 2020) and affinity-purification method (Gordon et al., 2020) could not identify RNF5 in the ORF3a

376 interactome although their selections contain comparable or more numbers of findings in the list. Thus, our

377 Spot-TurboID method is superior to previous methods for identifying proteins of interest in an interactome.

378 Since MAM is a cholesterol-enriched intracellular lipid raft (Area-Gomez et al., 2012), several proteins

379 associated with lipid metabolism and transport are observed at this domain (Kwak et al., 2020). Notably,

380 among 119 proteins identified in ORF3a interactome in this study, 34 proteins were earlier characterized as

381 lipid binding proteins (LBP) (Niphakis et al., 2015) and 15 proteins were reported at the MAM (Kwak et al.,

382 2020) (Dataset S1). Thus, we speculate that ORF3a forms neo-organelles (i.e. CM and DMV) by altering

383 ultrastructure and lipid contents of MAM since dynamic lipid transport at the MAM (Phillips and Voeltz, 2016)

384 can supply lipid molecules for the formation of autophagosomal membrane formations (Hamasaki et al., 2013).

385 Since several lysosome-associated membrane proteins (e.g. VPS16, RAB7A) are observed in our ORF3a

386 interactome as also observed in other ORF3a interactome list using affinity purification method (Stukalov et

387 al., 2021), consistent with recent reports that ORF3a is localized at the late endosome and lysosome (Miao et

388 al., 2021; Zhang et al., 2021), we postulated that these ORF3a-associated vesicles (e.g., CM and DMV) might

389 be interconnect or fused with the lysosome at some points.

390 Additionally, MAM is a site of active calcium transport from the ER to the mitochondria; a recent study

391 characterized ORF3a of SARS-CoV-2 as Ca2+ and K+ channels. Thus, we postulate that ORF3a may derange

392 the calcium transport, which in turn, can negatively impact MAM function. Since abnormal MAM formations

393 correlate with various metabolic diseases such as type 2 diabetes (Tubbs et al., 2018) and neurodegenerative

394 diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (Schon and

395 Przedborski, 2011), and Wolfram syndrome (Delprat et al., 2018), further studies might be required to monitor

396 the possible progression of these MAM-related metabolic diseases in SARS-CoV-2-infected patients (Rahman

15 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 397 et al., 2020).

398 In addition, we also found that many of the proteins in the ORF3a and M interactomes are targeted by

399 approved drug molecules, which may be useful for drug repurposing therapeutic strategies. The druggable

400 proteins targeted by previously approved drug molecules are listed in Supplementary Table 2-3.

401 In this study, we also provide evidence that our Spot-TurboID workflow using the GFP/GBP binding system

402 (vPOI-GFP/TurboID-GBP) is effective for interactome mapping of viral proteins in live cells. Since several

403 viral proteins are initially cloned with GFP for the imaging experiments typically performed in the initial

404 stages of many virus studies, we expect that our TurboID-GBP could be an effective “plug-and-playable”

405 component for rapid host interactome analysis with various GFP-tagged viral proteins, without cloning

406 additional constructs for PL experiments. This is especially relevant in situations where time is a constraint,

407 such as that in the current COVID-19 pandemic.

408 409

16 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 410 Resource Availability 411 Lead Contact 412 Further information and request of reagents or resources should be directed Contact, Dr. Rhee H. W. 413 ([email protected]) 414 415 Materials Availability 416 Upon reasonable requests, unique reagents utilized in this paper could be provided. 417 418 Data and Code Availability 419 The raw LC-MS data generated from this study have been deposited on ProteomeXchange Consortium 420 (accession no. PDX022355, User name: [email protected], Password: jvuXMLgS). 421 422 Materials

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies V5 Tag Monoclonal Antibody (mouse) Invitrogen #R960-25 GFP Monoclonal Antibody (mouse) Invitrogen #MA5-15256 Anti-StrepMAB-Classic, purified, lyophilized, ( IBA #2-1597-001 mouse) HA Tag Polyclonal Antibody Thermofisher #71-5500 FLAG rabbit Polyclonal Antibody Sigma aldrich #F7425 Ubiquitin Antibody Santa Cruz biotech #SC-166553 nology Alexa Fluor 568 IgG mouse Molecular probes #A11004 Streptavidin, Alexa Fluor 647 conjugate Invitrogen #S21374 Goat Anti-Mouse IgG (H + L)-HRP conjugate Bio-rad #1706516 Anti-rabbit IgG, HRP-linked Antibody Cell signaling #7074S Streptavidin-HRP Thermofisher #21126 Anti-Flag affinity gel beads Bimaker #B23102 4% paraformaldehyde CHEMBIO #CBPF-9004 HyClone Dulbecco’s Modified Eagle Medium Cytiva #SH30022.01 (DMEM) with high glucose: Liquid FBS Atlas Biologicals #EF-0500-A biotin Alfa aesar #A14207 doxycycline Sigma aldrich RIPA lysis buffer ELPISBIO #EBA-1149 Protease inhibitor cocktail Invitrogen #78438 Reagent for mass preparation Sodium azide Alfa aesar #14314 Sodium ascorbate Sigma aldrich #A4034 Trolox Sigma aldrich #238813 Urea Sigma aldrich #U5378 20X TBS Thermofisher #28358 Acetone Sigma aldrich #650501 Ammonium bicarbonate Sigma aldrich #A6141 Sodium dodecyl sulfate Sigma aldrich #436143 TPCK-Trypsin Thermofisher #20233 Dithiothreitol Sigma aldrich #43819 Iodoacetoamide Sigma aldrich #I1149 Trifluoroacetic acid Sigma aldrich #T6508-10AMP Formic acid Thermofisher #28905 17 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Streptavidin-coated magnetic beads Pierce #88817 Equipment Speed-vac Thermomixer 423 424 Methods 425 Cell lines 426 HEK293 cells and HEK293T cells were gift from Professor Kim, H. at Ulsan National Institute of Science 427 and Technology (Korea). SEC61B-V5-TurboID stably expressed HEK293 Flip-in T-rex cells were from the 428 past paper. HeLa cells and A549 cells were from Laboratory of Dr. Mun at Korea Brain Research Institute 429 (Korea). Cell lines were cultured according to the manufacturer’s instructions. 430 431 Expression Plasmids 432 Genes with epitope tag (i.e. V5, FLAG, HA, twin strep) were cloned into pCDNA3, pCDNA3.1, and pCDNA5 433 using digestion with enzymatic restriction sites and ligation with T4 DNA ligase. After digesting PCR products 434 and cut vectors simultaneously, they were ligated. CMV promoter made genes expressed possible in 435 mammalian cells. Table S1 summarized constructs information. Templates of SARS-CoV-2 were obtained 436 from Professor Kim, Ho Min (KAIST) and Professor Kim, V. Narry (SNU). 437 438 Cell Culture and Transfections 439 Cell lines were examined for morphology and confluence using a microscope. Cells were incubated with in 440 Dulbecco’s Modified Eagle Medium (Cytiva, cat. No., SH30022.01) supplemented with 10% fetal bovine 441 serum (Atlas biologicals, cat. No., EF-0500-A) in a 37 °C incubator 5% CO2 (v/v). Genes were introduced 442 into cells using PEI, when 60–80% cell confluence. 443 444 Stable Expression of SEC61B-V5-TurboID Cell Line (Flip-in HEK293T-Rex) 445 SEC61B-V5-TurboID expression was induced by treatment of 5 ng/mL doxycycline (Sigma Aldrich). For 446 transient expression of other constructs, transfection using PEI could do the same time with treatment of 447 doxycycline. 448 449 Biotin Labeling in Live Cells 450 Using transient transfection reagent, PEI, genes including TurboID or BioID were introduced into HEK293- 451 AD, HEK293 or HEK293T cells. After 16–24 h, the medium was changed to 500 μL (for 24 well plate) or 1 452 mL (for 12 well plate) of fresh growth medium containing 50 μM biotin (Alfa aesar, cat. No. A14207). These 453 cells were incubated at 37 °C and 5% CO2 for 30 min for using TurboID (or 16 h for using split BioID) in 454 accordance with the previously published protocols. Next, the reaction was stopped by washing, two times, 455 with Dulbecco's phosphate-buffered saline (DPBS). After, lysis was followed for western blot analysis or mass 456 sampling, or fixation was followed for fluorescence microscope imaging. 457 Using transient transfection reagent, PEI, genes including APEX2 were introduced into HEK293-AD, 458 HEK293 or HEK293T cells. After 16–24 h, the medium was changed to 500 μL (for 24 well plate) or 1 mL 459 (for 12 well plate) of fresh growth medium containing 250 μM desthiobiotin-phenol. These cells were 460 incubated at 37 °C and 5% CO2 for 30 min in accordance with the previously published protocols (Lee et al., 461 2017). H2O2 (1 mM; Sigma Aldrich, cat. No., STBJ2658) was added to each well for 1 min at room 462 temperature. Next, this reaction was terminated by washing, two times, with Dulbecco's phosphate-buffered 463 saline (DPBS) containing 5 mM Trolox (Sigma Aldrich, cat. No., 238813-25G), 10 mM sodium azide (Alfa 464 Aesar, cat. No., N07E043), and 10 mM sodium ascorbate (Sigma Aldrich, cat. No., A4034-100G). After, lysis 465 was followed for western blot analysis or mass sampling, or fixation was followed for fluorescence microscope 466 imaging. 467 468 Fluorescence Microscope Imaging 469 Cells were split on the coverslips (thickness no. 1.5 and radium: 18 mm) for microscope imaging.vPOI-GFP 470 expressed cells were fixed using 4% paraformaldehyde solution (CHEMBIO, cat. No. CBPF-9004) at room 471 temperature for 10–15 min. Cells were then washed with Dulbecco’s phosphate-buffered saline (DPBS) two 18 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 472 times. To detect vPOI-GFP expression, washed cells were maintained in DPBS on 4 °C refrigerator for 473 imaging by Leica (NICEM in Seoul National University, Korea) with objective lens (HC PL APO 100x/1.40 474 OIL), White Light Laser (WLL, 470–670 nm, 1 nm tunable laser), HyD detector and controlled by LAS X 475 software. 476 Co-expressed or biotin-labeled cells were fixed with 4% paraformaldehyde solution (CHEMBIO, cat. No. 477 CBPF-9004) in DPBS at room temperature for 10–15 min. Cells were then washed with DPBS, two times, 478 and permeabilized with cold methanol at -20 °C for 10 min. Cells were washed again, two times, with DPBS 479 and blocked for 30 min with 2% BSA in DPBS at room temperature (25°C). To detect expression pattern or 480 biotinylation pattern, the primary antibody, such as anti-V5 (Invitrogen, cat. No. R960-25, 1:5,000 dilution), 481 was incubated for 1 h at room temperature. After washing two times with TBST, cells were simultaneously 482 incubated with secondary Alexa Fluor 568 IgG mouse (Molecular probes, cat. No. A11004, 1:1,000 dilution) 483 and Streptavidin, Alexa Fluor 647 conjugate (Invitrogen, cat. No. S21374, 1:1,000 dilution) for 30 min at 484 room temperature. Cells were then washed two times with TBST and maintained in DPBS at 4 °C for imaging 485 using Leica (NICEM in Seoul National University, Korea). Pearson correlation was conducted using image J 486 software program (NIH). 487 488 Co-immunoprecipitation 489 Constructs were transfected using PEI. After more than 16 h, samples were lysed using RIPA buffer 490 (ELPISBIO, cat. No. EBA-1149) including 1× protease inhibitor cocktail (Invitrogen, cat. No. 78438) for 10 491 min at room temperature. After transferring these lysates to e-tube, samples were vortexed for 2–3 min at room 492 temperature. After centrifugation at 15,000 × g for 10 min at 4 °C, supernatant was collected. These samples 493 were incubated with already washed Anti-Flag affinity gel beads (Bimaker, cat. No.B23102) for 2 h at 4 °C. 494 Then they were washed more than three times and eluted using 1× SDS-PAGE loading buffer at 95 °C for 5 495 min. 496 497 Obtaining Secreted Proteins in Cell Culture Media 498 After biotin labeling of HEK293 cells expressing SEC61B-TurboID, cells were incubated with no FBS 499 DMEM for 16 h. After collecting these cell culture media in 15 mL tube, these samples were concentrated 500 using amicon filter (Millipore, cat. No. UFC501096) at 12,000 × g at 4 °C for 20 min. Concentrated proteins 501 were collected using 8 M urea (Sigma Aldrich, cat. No. U5378). These samples were used for immunoblotting. 502 503 Immunoblotting 504 Transfected and biotinylated cell samples were lysed using RIPA buffer (ELPISBIO, cat. No. EBA-1149) 505 including 1× protease inhibitor cocktail (Invitrogen, cat. No. 78438) for 10 min at room temperature. After 506 transferring these lysates to e-tube, samples were vortexed for 2–3 min at room temperature. Using 507 centrifugation at 15,000 × g for 10 min at 4 °C, samples were clarified. They were boiled with 1× SDS-PAGE 508 loading buffer at 95 °C for 5 min. After resolving protein samples using SDS-PAGE and transferring protein 509 samples to membrane, immunoblotting analysis was performed using antibodies. Membranes were blocked 510 for 30 min with 2% skim milk solution at room temperature. After washing three times with TBST for 5 min 511 each, at room temperature, primary antibodies, namely anti-V5 (Invitrogen, cat. No. R960-25, 1:10,000 512 dilution), anti-GFP (Invitrogen, cat. No. MA5-15256, 1:3,000 dilution), anti-HA (Thermofisher, cat. No. 71- 513 5500, 1:10,000 dilution), anti-FLAG (Sigma Aldrich, cat. No. F7425, 1:10,000 dilution), and anti-strep (IBA, 514 cat. No. 2-1597-001, 1:1,000 dilution), was incubated for 16 h at 4 °C. After washing three times with TBST 515 for 5 min at room temperature, secondary antibodies, namely mouse-HRP (Bio-rad, cat. No. 1706516, 1:3,000 516 dilution) and rabbit-HRP (Cell signaling, cat. No. 7074S, 1:3,000 dilution), or SA-HRP (Thermofisher, cat. 517 No. 21126, 1:10,000 dilution) were incubated for 1 h at room temperature. After washing three times with 518 TBST for 5 min at room temperature, results of immunoblotting assay were obtained using ECL solution using 519 GENESYS program. Line scan analysis was performed using image J software program (NIH). After 520 subtraction of intensity value at background, protein bands from top to bottom of each lane were putted as x- 521 axis and intensity of bands from top to bottom of each lane was putted as y-axis. 522 523 EM imaging and CLEM 524 To observe the DAB-stained cells, cells were cultured in 35-mm glass grid-bottomed culture dishes (MatTek

19 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 525 life sciences, MA, USA) to 30–40% confluency. Then cells were transfected with DNA plasmids using 526 Lipofectamin 2000 (Life Technologies, Carlsbad, CA, USA). Next day, cells were fixed with 1% 527 glutaraldehyde (Electron Microscopy Sciences, cat. No. 16200) and 1% paraformaldehyde (Electron 528 Microscopy Sciences, cat. No. 19210) in 0.1 M cacodylate solution (pH 7.0) for 1 h at 4 ℃. After washing, 529 20 mM glycine solution was used for quench unreacted aldehyde. DAB staining (DAB, Sigma, cat. No. D8001) 530 took approximately 20−40 min until a light brown stain was visible under an inverted light microscope. DAB 531 stained cells were post-fixed with 2% osmium tetroxide in distilled water for 30 min at 4 °C and en bloc in 1% 532 uranyl acetate (EMS, USA, cat. No. 22400) overnight and dehydrated with a graded ethanol series. The 533 samples were then embedded with an EMBed-812 embedding kit (Electron Microscopy Sciences, USA, cat. 534 No. 14120) and polymerized in oven at 60 ℃. The polymerized samples were sectioned (60 nm) with an 535 ultramicrotome (UC7; Leica Microsystems, Germany), and the sections were mounted on copper slot grids 536 with a specimen support film. Sections were stained with uranyless (Electron Microscopy Sciences, cat. No. 537 22409) and lead citrate (Electron Microscopy Sciences, cat. No. 22410) then viewed on a Tecnai G2 538 transmission electron microscope (ThermoFisher, USA). 539 For the CLEM observation, the ORF3a-GFP, M-mCherry and M-GFP transfected cells were imaged under 540 a confocal light microscope (Ti-RCP, Nikon, Japan) in living cell, and cells were fixed with 1% glutaraldehyde 541 and 1% paraformaldehyde in 0.1 M cacodylate solution (pH 7.0). After washing, cells were dehydrated with 542 a graded ethanol series and infiltrated with an embedding medium. Following embedment, 60 nm sections 543 were cut horizontally to the plane of the block (UC7; Leica Microsystems, Germany) and were mounted on 544 copper slot grids with a specimen support film. Sections were stained with uranyl acetate and lead citrate. The 545 cells were observed at 120 kV in a Tecnai G2 microscope (ThermoFisher, USA). Confocal micrographs were 546 produced as high-quality large images using PhotoZoom Pro 8 software (Benvista Ltd., Houston, TX, USA). 547 Enlarged fluorescence images were fitted to the electron micrographs using the Image J BigWarp program. 548 549 Proteome Digestion & Enrichment of Biotinylated Peptides 550 For mass sampling, HEK293T cells were split in three T75 flasks for triplicate samples per each condition. 551 For transiently co-expressed two constructs, vPOI-GFP and TurboID-GBP, cells were grown at 70–80% 552 confluence and transfected with wanted constructs using PEI. After 16 h (transfection), 50 μM biotin (Alfa 553 aesar, cat. No. A14207) were treated for 30 min in 37 °C incubator. After biotin labeling, these cells were 554 washed 3–4 times with cold DPBS and lysed with 1.5 mL 2% SDS in 1× TBS (25 mM Tris, 0.15 M NaCl, pH 555 7.2, Thermoscientific, cat. No. 28358) containing 1× protease inhibitor cocktail. These lysates were underwent 556 ultrasonication (Bioruptor, diagenode) 3–4 times for 15 min in a cold room. For removing free biotin, 5–6 557 times the sample volume of cold acetone stored at -20 °C (Sigma Aldrich, cat. No. 650501) was mixed with 558 lysates and stored at -20 °C for least 2 h. These samples were centrifuged at 13,000 × g for 10 min at 4 °C. 559 Supernatant solution was gently discarded. Cold acetone, 6.3 mL, and 700 μL of 1× TBS were mixed with the 560 pellet. These mixtures were then vigorously vortexed and stored at -20 °C for least 2 h or overnight. The 561 samples were then centrifuged at 13,000 × g for an additional 10 min at 4 °C. Supernatant solution was gently 562 discarded. After pellet was naturally dried for 3–5 min, it was resolubilized with 1 mL of 8 M urea (Sigma 563 Aldrich, cat. No. U5378) in 50 mM ammonium bicarbonate (ABC, Sigma Aldrich, cat. No. A16141). Protein 564 concentration was calculated using the BCA assay. Samples were vortexed at 650 rpm for 1 h at 37 °C using 565 Thermomixer (Eppendorf) for denaturation. Samples were reduced in 10 mM dithiothreitol (Sigma Aldrich, 566 cat. No. 43816) at 650 rpm for 1 h at 37 °C using Thermomixer (Eppendorf), while samples alkylation was 567 accomplished in 40 mM iodoacetoamide (Sigma Aldrich, cat. No. I1149) at 650 rpm for 1 h at 37 °C using 568 Thermomixer (Eppendorf). These samples were diluted 8 times with 50 mM ABC (Sigma Aldrich, cat. No. 569 A16141). CaCl2 (Alfa aesar, cat. No. 12312) was added for the final concentration of 1 mM. Samples were 570 digested using trypsin (Thermoscientific, cat. No. 20233) (50:1 w/w) at 650 rpm for 6–18 h at 37 °C using 571 Thermomixer (Eppendorf). Insoluble materials were removed using centrifugation for 3 min at 10,000 × g. 572 Streptavidin beads (300 μL; Pierce, cat. No. 88817) were washed using 2 M urea in 1× TBS for 3–4 times, 573 were then added to samples, and then rotated for 1 h at room temperature. The flow-through fraction was not 574 discarded, and beads were washed 2–3 times using 2 M urea in 50 mM ABC. After removing the supernatants, 575 beads were washed using pure water and transferred to new tubes. After adding 500 μL 80% acetonitrile 576 (Sigma Aldrich, cat. No. 900667), 0.2% TFA (Sigma Aldrich, cat. No. T6508) and 0.1% formic acid (FA, 577 Thermoscientific, cat. No. 28905), heat such biotinylated peptides at 60 °C and mixed at 650 rpm. Supernatant 20 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 578 without streptavidin beads was transferred to new tubes. This elution step was repeated at least 4 times. These 579 total elution fractions were dried for 5 h using Speed-vac (Eppendorf). Before samples were injected to mass 580 spectrometry, they were stored at -20 °C. 581 582 Calculation of Similarity Value 583 Similarity value of each mass samples was calculated as previously reported. The correlation index, Cij, was 584 introduced for calculation of similarity value. The Equation below shows how similarity value was 585 calculated. 1 푁 ∑푘=1 퐼푖(푘)퐼푗(푘) 586 퐶 = 푁 푖푗 1 1 √ ∑푁 퐼 (푘)2 √ ∑푁 퐼 (푘)2 푁 푘=1 푖 푁 푘=1 푗 587 As the result of Cij covers from 0 to 1, bigger value indicates more similarity, while smaller value indicates 588 less similarity. 589 590 LC-MS/MS Analysis of Enriched Peptide Samples 591 Analytical capillary columns (100 cm × 75 μm i.d.) and trap columns were packed in-house with 3 m Jupiter 592 C18 particles (Phenomenex, Torrance). The long analytical column was placed in a column heater (Analytical 593 Sales and Services) regulated to a temperature of 45 °C. NanoAcquity UPLC system (Waters, Milford) was 594 operated at a flow rate of 300 nL/min over 2 h with linear gradient ranging from 95% solvent A (H2O with 595 0.1% formic acid) to 40% of solvent B (acetonitrile with 0.1% formic acid). The enriched samples were 596 analyzed using Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) equipped with an in-house 597 customized nanoelectrospray ion source. Precursor ions were acquired (m/z 300–1,500) at 120 K resolving 598 power and the isolation of precursor for MS/MS analysis was conducted with a 1.4 Th. Higher-energy 599 collisional dissociation (HCD) with 30% collision energy was used for sequencing with auto gain control 600 (AGC) target of 1E5. Resolving power for acquired MS2 spectra was set to 30 k at with 200 ms maximum 601 injection time. 602 603 Processing MS data and Identification of Proteins 604 All MS/MS data were searched using MaxQuant (version 1.5.3.30) with Andromeda search engine at 10 ppm 605 precursor ion mass tolerance against the SwissProt Homo sapiens proteome database (20,199 entries, UniProt 606 (http://www.uniprot.org/)). The label free quantification (LFQ) and Match Between Runs were used with the 607 following search parameters: Semi-trypic digestion, fixed carbaminomethylation on cysteine, dynamic 608 oxidation of methionine, protein N-terminal acetylation with biotin labels of lysine residue. Less than 1% of 609 false discovery rate (FDR) was obtained for unique labeled peptide and as well as unique labeled protein. LFQ 610 intensity values were log-transformed for further analysis and missing values were filled by imputed values 611 representing a normal distribution around the detection limit. The imputation of protein intensity were 612 conducted using Perseus software program. 613 614 ACKNOWLEDGMENTS 615 This work was supported by the National Research Foundation of Korea (NRF-2020K1A3A1A47110634, 616 NRF-2019R1A2C3008463), the Organelle Network Research Center (NRF-2017R1A5A1015366) and a grant 617 of the Korea Health Industry Development Institute(KHIDI) funded by the Ministry of Health & Welfare and 618 Ministry of science and ICT, Republic of Korea (grant number : HU20C0326). H.W.R. was supported by 619 Creative-Pioneering Researchers Program through Seoul National University. J.Y.M. and M.J. are supported 620 by KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and 621 ICT (Information & Communication Technology) (20-BR-01-09). EM data were acquired at Brain Research 622 Core Facilities in KBRI. J.-S.K. are supported by IBS-R008-D1 of the Institute for Basic Science from the 623 Ministry of Science and ICT of Korea. 624 625 AUTHOR CONTRIBUTIONS 626 Y.B.L., J.Y.M., J.S.K., and H.W.R. conceived the project. M.J. and J.Y.M. contributed to EM imaging. J.K. 627 and J.S.K. performed LC-MS/MS experiment and mass data processing. M.G.K. contributed to confocal 628 imaging. C.K. contributed to preparation of mass samples. Y.B.L., J.Y.M., J.S.K., and H.W.R. wrote and edited

21 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 629 the manuscript. 630 631 DECLARATION OF INTEREST 632 The authors have no conflicting financial interests. A patent application relating to the interactome findings 633 has been filed by Seoul National University. 634 635 SUPPLEMENTAL INFORMATION 636 Table S1-3. 637 Figures S1-S15. 638 Data S1. Result from mass analysis of ORF3a and M interactome 639

22 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 640 FIGURE LEGENDS 641 642 Figure 1. Confocal imaging of GFP-tagged vPOI of SARS-CoV-2 (a) Schematic view of viral protein of 643 interest (vPOI)-linker-GFP constructs (linker sequence: GAPGSAGSAAGSG). Predicted transmembrane 644 (TM) domains by TMHMM program are shown in light gray. (b) Confocal microscopy results of vPOI-GFP 645 constructs in HEK293-AD cells. Scale bars: 10 μm. (c) Confocal microscopy images of co-expressed N-GFP 646 and G3BP1-V5. G3BP1-V5 was visualized using anti-V5 antibody (AF568-conjugated, red fluorescence 647 channel). Pearson correlation values were calculated between different fluorescent channel images. Scale bars: 648 8 μm (upper) and 10 μm (below). BF: bright field. 649 650 Figure 2. Interactome mapping of SARS-CoV-2 viral proteins by Spot-TurboID workflow (a) Overview 651 of our Spot-TurboID method combined with vPOI-GFP and TurboID-GBP binding system (b) Confocal 652 microscopy images of vPOI-GFP and TurboID-V5-GBP in HEK293-AD cells. TurboID-V5-GBP was 653 visualized by anti-V5 antibody (AF568-conjugated, RFP fluorescence channel). Biotinylated proteins were 654 visualized by AF647-conjugated streptavidin (Cy5 fluorescence channel). Scale bars: 10 μm. Additional 655 images are shown in Figure S2. BF: bright field. (c) Streptavidin-horseradish peroxidase (SA-HRP) western 656 blotting results of biotinylated proteins by TurboID-GBP under the co-expression of vPOI-GFP in HEK293T 657 cells. Band signals of biotinylated vPOI-GFP by TurboID-GBP are marked with green asterisks. Anti-V5 blot 658 signals of the same samples are shown below. (d) Anti-GFP western blotting results of the same samples as 659 (c). Bands of the expected molecular weight of each vPOI-GFP constructs are marked with green asterisks. 660 Raw images of blot results are shown in Figure S3. 661 662 Figure 3. EM results of ORF3a and Membrane protein of SARS-CoV-2 expressed cells (a) Schematic 663 view of ORF3a-linker-V5-APEX2 and M-linker-V5-APEX2 constructs. Linker sequence: 664 GAPGSAGSAAGSG. TM domains of vPOI are shown in light gray. (b) Proposed membrane topology of 665 ORF3a-APEX2 and M-APEX2 at the ER membrane (c) EM images of DAB-stained ORF3a-APEX2- and 666 ORF3a-GFP-transfected HeLa cells. Scale bars: 1 μm. Distinct membrane structures were observed with 667 higher magnification. Magnified areas are marked with different colored boxes. Additional EM images are 668 shown in Figure S5a. Parts with red arrows mark DMV structures (c, e, g). Mitochondria and cubic membrane 669 are indicated as “m” and “CM”, respectively (c–g). (d) EM images of M-APEX2 transfected HeLa cells. Scale 670 bars: 1 μm. Additional EM images are shown in Figure S5b. (e) EM images of ORF3a-APEX2 transfected 671 A549 cells. Scale bas: 500 nm–2 μm. (f) EM images of M-APEX2 transfected A549 cells. Scale bars: 1 μm– 672 2 μm. (g) CLEM images of ORF3a-GFP and M-mCherry co-transfected A549 cells. Ultrastructures of 673 endomembrane systems in each cell were observed by EM imaging. Scale bars: 500 nm–5 μm. 674 675 Figure 4. Mass analysis of host interactome of ORF3a and M protein of SARS-CoV-2 by Spot-TurboID 676 (a) Schematic view of three groups of sample for mass analysis: TurboID-GBP + GFP (left, control), TurboID- 677 GBP + ORF3a-GFP (middle), and TurboID-GBP + M-GFP (right). (b) Enrichment of the Spot-TurboID 678 detected biotinylated proteins by TurboID-GBP in the samples of ORF3a-GFP (upper) or M-GFP (below) over 679 GFP (control), respectively. x-axis: Log2 value of fold change (FC) of mass signal intensities. cut off value is 680 2.3; y-axis: -Log10 value of p-value, cut off value is 2.3. Gene names of the top 30 proteins are shown in the 681 graph. Bubble size indicated the mass signal intensity. See detailed information in Figure S7e and Dataset 682 S1. (c) Subcellular distribution of the selected interactome of ORF3a (117 proteins) and M (191 proteins). See 683 detailed information in Figure S7f and Dataset S1. (d) Gene ontology analysis 684 (http://bioinformatics.sdstate.edu/go/) of enriched functions of ORF3a and M’s interactomes. See detailed 685 information in Figure S7g. (e, f) Subcellular map of top 30 selected interactome of ORF3a (e) and M (f). 686 MAM proteins are shown in yellow-colored box. 687

23 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 688 Figure 5. Proposed membrane topologies of the integral membrane proteins in ORF3a and M’s 689 interactome list. Biotin-labeled sites by TurboID-GBP (cytosolic side) were colored according to their 690 samples (red: observed in ORF3-GFP samples; blue: observed in M-GFP samples; purple: observed both in 691 ORF3a and in M samples). Newly proposed membrane topologies are shown in yellow. Detailed information 692 is given in Figure S8 and Dataset S1. 693 694 Figure 6. RNF5 ubiquitinylate ORF3a of SARS-CoV-2. (a) Highlighted RNF5 in ORF3a interactome (left) 695 and in M interactome (right) volcano plots shown in Figure 4b. (b) Confocal microscopy images of Flag- 696 RNF5 in HEK293-AD cells. Flag-RNF5 was visualized by anti-Flag antibody (AF488-conjugated, GFP 697 fluorescence channel). Scale bars: 10 μm. (c) Confocal microscopy images of vPOI-GFP with Flag-RNF5. 698 Flag-RNF5 was visualized by anti-Flag antibody (AF647-conjugated, Cy5 fluorescence channel). BF: bright 699 field, Scale bars: 10 μm. (d) Western blot analysis of ORF3a ubiquitination by RNF5 in HEK293T cells. Anti- 700 Flag and Anti-V5 were utilized for ORF3a-Flag and V5-UBB, respectively. Red arrow marks the unmodified 701 molecular weight of ORF3a-Flag. Blue arrows mark ubiquitinylated ORF3a-Flag. Same molecular weight 702 bands were detected in both anti-Flag and anti-V5 blot results. Additional blot results are shown in Figure 703 S9d-e and Figure S10. (e) Proposed model of RNF5-mediated ubiquitination of ORF3a of SARS-CoV-2. Zinc 704 finger domain of RNF5 (27–68 aa) is highlighted with yellow box, and 42 aa is an essential site for activity of 705 E3 ubiquitin ligase. 706 707 Figure 7. ORF3a remodeled local proteome at the MAM. (a) Local protein overlaps between interactomes 708 of ORF3a (117 proteins), M (191 proteins), and MAM proteome (115 proteins). (b) Pie chart of functionally 709 classified 28 overlapped proteins between ORF3a interactome and MAM proteome. (c) Pie chart of 710 functionally classified 24 overlapped proteins between M interactome and MAM proteome. (d) Scheme of 711 Contact-ID assay to check the MAM proteome changes by ORF3a expression. (e) SA-HRP western blotting 712 results of Contact-ID with or without ORF3a co-expression. Biotinylated ORF3a (lane 4, 5) are marked with 713 red arrow. Additional biotinylated proteins (lane 4, 5) are marked with blue arrows. Raw images are shown in 714 Figure S11. (f) Scheme of in situ biotinylation of secreted protein (iSLET) by SEC61B-TurboID. (g) SA-HRP 715 western blotting results of secreted biotinylated proteins from SEC61B-TurboID expressed HEK293 cells with 716 or without co-expression of ORF3a. Raw images are shown in Figure S13a. (h) Anti-strep western blot results 717 of the same samples as (b). Bands of ORF3a-strep (lane 4, 5) are marked with a red arrow. Raw images are 718 shown in Figure S13c. 719 720

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Dev Cell 56, 427-442 e425. 834 Monel, B., Compton, A.A., Bruel, T., Amraoui, S., Burlaud-Gaillard, J., Roy, N., Guivel-Benhassine, F., Porrot, 835 F., Génin, P., Meertens, L., et al. (2017). Zika virus induces massive cytoplasmic vacuolization and paraptosis- 836 like death in infected cells. EMBO J 36, 1653-1668. 837 Nii, S., Morgan, C., and Rose, H.M. (1968). Electron microscopy of herpes simplex virus. II. Sequence of 838 development. J Virol 2, 517-536. 839 Niphakis, M.J., Lum, K.M., Cognetta, A.B., 3rd, Correia, B.E., Ichu, T.-A., Olucha, J., Brown, S.J., Kundu, 840 S., Piscitelli, F., Rosen, H., et al. (2015). A Global Map of Lipid-Binding Proteins and Their Ligandability in 841 Cells. Cell 161, 1668-1680. 842 Nover, L., Scharf, K.D., and Neumann, D. (1983). Formation of cytoplasmic heat shock granules in tomato 843 cell cultures and leaves. Mol Cell Biol 3, 1648-1655. 844 Oshiumi, H., Mifsud, E.J., and Daito, T. (2015). 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28 1 84 1 238 1 275 1 238 Nsp7-GFP ORF3a-GFP A bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446555; this version posted June 1, 2021. The copyright holder for this preprint Figure 1 (which was not certified1 by199 peer 1review)238 is the author/funder, who has granted bioRxiv a license to display the preprint in1 perpetuity.22 1 238 It is Nsp8-GFP made available under aCC-BY-NC 4.0 International license. ORF3b-GFP 1 61 1 238

structural 1 114 1 238 - protein Nsp9-GFP ORF6-GFP

Non 1 299 1 238 1 121 1 238 Nsp16-GFP ORF7a-GFP

1 43 1 238 1 1273 1 238 ORF7b-GFP S-GFP 1 121 1 238 1 75 1 238 ORF8-GFP E-GFP

Accessory protein Accessory 1 97 1 238 1 222 1 238 ORF9b-GFP

protein M-GFP

Structural Structural 1 73 1 238 238 ORF9c-GFP N-GFP 1 419 1 1 38 1 238 ORF10-GFP B GFP BF Merged GFP BF Merged

Nsp7-GFP ORF3a-GFP

Nsp8-GFP ORF3b-GFP structural protein - Non Nsp9-GFP ORF6-GFP

M-GFP ORF7b-GFP Accessory protein

N-GFP ORF9b-GFP Structural protein

ORF9c-GFP

C N-GFP G3BP1-V5 BF Merged

V5 Pearson R - = 0.82 G3BP1 +

Pearson R = 0.82 GFP GFP - N A Identification of proteins interacting with vPOI using GFP-GBP system Figure 2

13 aa linker : Biotinylation vPOI GAPGSAGSAAGSG TurboID B V5 Co-transfection GFP (30 min) vPOI

B

+ GBP GBP GFP

SA bead Trypsin Mass enrichment digestion analysis B B vPOI Conventional B method B B B Cell Lysis True False

B B B Spot-TurboID

(Our method) B Trypsin B SA bead B Mass digestion enrichment analysis Lys + 226 Da True (biotin-PTM) (>99%) V5 SA BF Merged (V5 + SA) Merged B V5 SA GFP BF (V5 + GFP)

TurboID-V5-GBP Pearson R TurboID-V5-GBP = 0.85 + ORF3a-GFP Pearson R Merged = 0.80 V5 SA GFP BF (V5 + GFP)

TurboID-V5-GBP TurboID-V5-GBP + M-GFP + ORF6-GFP Pearson R Pearson R = 0.91 = 0.88 Accessory protein Accessory

TurboID-V5-GBP TurboID-V5-GBP

Structuralprotein + N-GFP + ORF7b-GFP Pearson R Pearson R = 0.95 = 0.92 C TurboID-V5-GBP TurboID-V5-GBP TurboID-V5-GBP

GFP GFP GFP GFP GFP GFP GFP GFP GFP GFP ------GFP - GFP - - GFP GFP GFP GFP ------

GFP GFP GFP GFP GFP GFP GFP - GFP ------ctrl nsp16 S ORF3a E M ORF6 ORF7a ORF7b ORF8 N ctrl nsp16 S ORF3a E M ORF6 ORF7a ORF7b ORF8 N ctrl ORF3b ORF9b ORF9c ORF10 Biotin + + + + + + + + + + + + + + + + + + + + + + Biotin + + + + +

Lane number 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 Lane number 1 12 13 14 15

kDa kDa 245 245 180 130 180 130 100 100 75 75

63 63

48 48

35 35

25 25

WB : SA-HRP (short exposure) WB : SA-HRP (long exposure) WB : SA-HRP

Lane number 1 2 3 4 5 6 7 8 9 10 11 WB : Anti-V5 D WB : Anti-V5 (for TurboID-V5-GBP) 1 12 13 14 15 Lane number 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 Lane number

kDa kDa

245 180 180 130 130

100 100 75 75

63 63

48 48

35 35

25

25

WB : Anti-GFP (short exposure) WB : Anti-GFP (long exposure) WB : Anti-GFP A B Figure 3 ORF3a-APEX2 Membrane-APEX2

N N 1 34 56 77 99103 125 275 1 249 V5 APEX2 ORF3a-V5-APEX2 ER lumen 34 99 103 20 73 78 ER membrane

1 20 39 51 73 78 100 222 1 249 cytosol 56 77 125 39 51 100 V5 APEX2 M-V5-APEX2

C C

C HeLa : ORF3a-APEX2 ORF3a-GFP D HeLa : M-APEX2

m DMV

m m m m m m MLB CM

m DMV m m m m m m CM m

E A549 : ORF3a-APEX2 F A549 : M-APEX2

m MLB m ER DMV m m ER CM

m DMV

m

ER Golgi CM

G A549 : ORF3a-GFP + M-mCherry

Cell (1) CM Cell (2)

ORF3a-GFP

M-mCherry CM DMV

m

CM ER Cell (1) m DMV Merged Cell (2) m ER

CM m A Ctrl : GFP + TurboID-GBP ORF3a-GFP + TurboID-GBP M-GFP + TurboID-GBP Figure 4

B B B ORF3a M

B B

GBP GFP GBP GFP GBP GFP B C Among 117 proteins, Top 30 interactome selected as intensity of mass signal

GBP 47 44 21 14 9 - TMEM165 ORF3a 5 C1orf21 TMEM110 SLC22A5 CLCC1 EPHA2 BET1 SCD5 95 48 4 28 16 4 ZC3HAV1 M TMX1 SAR1A TurboID UBXN8 VAMP3

+ BET1L DHCR7 0% 50 % 10 0% RNF5 Log p VMA21 3 PTPN1 ITGB1 - SLC16A1 plasma membrane endom embra ne mitochondria ERC2 117 proteins selected FKBP8 UBXN4 peroxisome nucleus/cytosol others

GFP SCD NEDD1 - as ORF3a interactome EPB41L2 JAM3 ADAM9 SEC63 SEC61B Log 2 FC ≥ 2.3 2 2 3 4 5 6 7 ￚ Log 10 p-value ≥ 2.3

ORF3a Log 2 FC D Among 191 proteins, Top 30 interactome selected as intensity of mass signal ORF3a GO analysis Enrichment M GO analysis Enrichment FDR FDR

Regulation of localization GBP 2.89E-05 Protein - 6 VMA21 ITGB1 8.11E-12 GLIPR2 localization TMEM110 Ubiquitin-dependent UBIAD1 CISD2 MCAM 5 JAM3 0.000204 Nitrogen compound ERAD pathway 1.35E-11 ESYT2 VAMP3 SLC3A2 transport EPB41L2 LSR Regulation of sterol TurboID 4 SLC7A3 BET1L UBR3 0.000431 Organic substance WDR6 SLC16A1 biosynthetic process 1.45E-11 Log p ERC2 CXADR transport

- 3 SLC22A5 191 proteins selected IRS4 STEAP3 Regulation of cholesterol PPFIA1 TMEM2 0.000431 Establishment of SAR1A biosynthetic process 3.45E-11 GFP + as M interactome YKT6 SLC29A1 protein localization - 2 DSG2 LBR 2

M Log FC ≥ 2.3 2 3 4 5 6 7 Protein exit from Macromolecule ￚ 10 endoplasmic reticulum 0.000431 1.31E-10 Log p-value ≥ 2.3 Log 2 FC localization

SLC16A1 SLC22A5 E F SLC16A1 SLC22A5 TMEM2 UBR3 VAMP3 JAM3 TMEM110 TMEM165 MCAM ESYT2 LSR TMEM110 BET1L EPHA2 BET1L PTPN1 CXADR UBXN8 GLIPR2 SCD EPB41L2 EPB41L2 STEAP3

TMX1 SEC63 ORF3a CLCC1 DSG2 M ADAM9 DHCR7 LBR UBXN4 IRS4 SCD5 VMA21 VAMP3 VMA21 ITGB1 ITGB1 JAM3 ZC3HAV1 RNF5 SEC61B UBIAD1 SAR1A SLC29A1 SAR1A ERC2 C1of21 ERC2 BET1 CISD2 YKT6 NEDD1 FKBP8 SLC7A3 SLC3A2 PPFIA1 WDR6

Protein transport and chaperone Autophagy/quality control Redox reaction Neuronal activity Others

Vesicle transport Protein degradation Ion transport Nucleic acid binding and process Unknown

Proteins characterized as interacting with Membrane integrity Cell cycle/apoptosis Lipid synthesis and steroid binding/process Antiviral/immune signaling viral proteins N N Figure 5

UBXN8 B B SCD5 (K104, K154, K160) C C (K10, K17) ADAM9 EPHA2 N (K781) (K578) N

B C B N B B B C N C B

B TMEM165 CLCC1 BB (K208) C

(K414, K437, K463, BB B B B

C

K470, K472, K518) B N

SCD B (K196, K338, K341)

N B DHCR7

(K11, K13) C B N B N TMX1 C B (K222, K223, B C B B JAM3 K233, K259) (K287, K305) C B BET1 B N B (K42, K47, B K58, K62) N SEC63 ORF3a (K534, K535) N B B C C B SLC16A1 B B SEC61B N C (K211, K216, K219, B (K20) K224, K473, K479) B B N C B

RNF5 C (K75) B B B C B N B TMEM110 N (K281) BET1L C

N (K31, K36) B

N

B VMA21 B

N FKBP8 B

C (K6) (K272, K314, K334, ITGB1 K340) (K784, K794) N B SLC22A5 C B (K553)

CISD2 B B C B C

(K95, K105, K131, N B B K132) LBR (K123, K147, K178,

K186, K190, K524) M VAMP3

B N

B (K35, K42)

B C

B B B N B

B

C STEAP3

(K7, K484) C B N

N B B C N UBIAD1 B

SLC29A1 B (K10) (K249, K255, K263) B C C B B B B B B B B B B ESYT2 N SLC3A2 (K405, K750) N B B

DSG2 (K140, K145, K147, C N LSR (K697, K706, K746, K160, K166) CXADR MCAM

K779, K832, K834, (K410, K638) (K616, K640)

UBR3 (K339) N B N

K844, K901, K919, B SLC7A3 C C TMEM2 B (K36) C C (K605) N K1109) B B B (K31, K48) B B B B B

B B B

N N

C C N C Figure 6 A 6 8 GBP - 5

GBP 6 -

4 RNF5 TurboID 4 + Log p Log p RNF5 TurboID - 3 - GFP - 2 GFP +

2 - 2 4 6 8 2 4 6 8 M ORF3a Log 2 FC Log 2 FC

B Flag BF C GFP Flag BF Merged + GFP - RNF5 - Flag ORF3a RNF5 - + Flag RNF5 - GFP - M Flag

HEK293T D : Co-IP using Flag-bead IP FT Elu

ORF3a-Flag ￚ + + + + + + + + + + + + 1 2 3 4 5 ￚ + + + ￚ + + + ￚ + + + ￚ Empty vector + + + WB : Anti-HA ￚ ￚ ￚ + + ￚ ￚ + + ￚ ￚ + + HA-RNF5 (for HA-RNF5) V5-UBB ￚ ￚ + ￚ + ￚ + ￚ + ￚ + ￚ +

Lane number 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13

kDa kDa 245 245 180 180 130 130 100 100 75 75

63 63

48 48

35 35

25 25

20 20

17 17

WB : Anti-Flag WB : Anti-V5 E (for ORF3a-Flag) (for V5-Ubiquitin B) SARS-CoV-2 viral infection

Ub N C 27 Ub 42 68 Ub RNF5 ORF3a Ub ORF3a Figure 7 C1orf43 TP53I11 A B MACO1 VMA21, SEC61B MAM proteome DNAJC1, STT3B : 115 proteins AGPAT1, ORF3a interactome interactome (Kwak et al. 2020) SCD5, SC5D, : 117 proteins DHCR7 SAR1A, BET1, Protein transport and chaperone USE1, SAR1B, PRAF2 CLCC1, Vesicle transport MMGT1 Membrane integrity 10 SEC63, 27 81 TMX1, HMOX2 LNPK ITGB1, Autophagy/quality control FKBP8, RNF5, UBXN4, overlapped with proteomeMAM 18 ORF3a : proteins 28 EMC6 UBE2J1 Protein degradation 62 6 Cell cycle/apoptosis

C1orf43, C Redox reaction TP53I11 VMA21, DNAJC1, 105 Ion transport SCD5, SERP1 SC5D, DHCR7 Lipid synthesis and steroid binding/process PRAF2, USE1 interactome MMGT1 SAR1A, SAR1B Neuronal activity TMX1, HMOX2, Others POR LBR, M interactome ZDHHC5, : 191 proteins LNPK

24 proteins : M M : proteins 24 ITGB1 RNF5, TMEM9B, overlapped with proteomeMAM UBE2J1 CISD2

MAM

D ER Outer Contact-ID membrane mitochondrial B membrane BioID(N term-78aa) BioID(79aa-C term) B

MAM *

TOM20 Normalcondition

ER Outer SEC61B membrane mitochondrial B membrane * MAM : Mitochondrial-Associated Membrane B

B B : ORF3a expression

B

HEK293T HEK293T E Lane number 1 2 3 4 5 TOM20-BioID(C-term) ￚ + + + + ￚ + + + + ￚ + + + + ￚ + + + + BioID(N-term)-SEC61B WB : Anti-HA for TOM20-BioID(C-term) ORF3a-strep ￚ ￚ ￚ + + ￚ ￚ ￚ + +

Empty vector ￚ + + ￚ ￚ ￚ + + ￚ ￚ WB : Anti-FLAG for BioID(N-term)-SEC61B Bitoin + + + + + + + + + +

Lane number 1 2 3 4 5 1 2 3 4 5 Lane number 1 2 3 4 5

kDa kDa 245 245 180 180 130 130

100 100 75 75

63 63

48 48

35 35

25 25

WB : SA-HRP WB : SA-HRP WB : Anti-strep (short exposure) (long exposure) (for ORF3a-Strep) Figure 7 (Continued) F Identification of secreted proteins using SEC61B-TurboID (iSLET) ER-lumen

B Collecting culture Protein B Western blot B media including concentration analysis B secreted proteins Using Amicon filter

Culture B expression Normalcondition media Normal condition vPOI SZ 1 2 3 4 ER-lumen B B

B B B B

B B B B WB : SA-HRP expression ORF3a or M B

HEK293 HEK293 HEK293 (transfected) (transfected) (transfected) H SEC61B-TurboID ￚ + + + + SEC61B-TurboID ￚ + + + + ￚ + + + + G ORF3a-strep ￚ ￚ ￚ + + ORF3a-strep ￚ ￚ ￚ + + ￚ ￚ ￚ + + Empty vector ￚ + + ￚ ￚ Empty vector ￚ + + ￚ ￚ ￚ + + ￚ ￚ Biotin + + + + + Biotin + + + + + + + + + + Lane number 1 2 3 4 5 Lane number 1 2 3 4 5 1 2 3 4 5 kDa kDa 245 245 180 180 130 130

100 100 75 75 Secreted proteins Secreted 63 63

Secreted proteins Secreted 48 48 35

35 25

WB : SA-HRP WB : SA-HRP 25 (short exposure) (long exposure) WB : Anti-strep (for ORF3a-Strep)