key: cord-1039002-dox32hb7
authors: Tabata, Keisuke; Prasad, Vibhu; Paul, David; Lee, Ji-Young; Pham, Minh-Tu; Twu, Woan-Ing; Neufeldt, Christopher J.; Cortese, Mirko; Cerikan, Berati; Si Tran, Cong; Lüchtenborg, Christian; V’kovski, Philip; Hörmann, Katrin; Müller, André C.; Zitzmann, Carolin; Haselmann, Uta; Beneke, Jürgen; Kaderali, Lars; Erfle, Holger; Thiel, Volker; Lohmann, Volker; Superti-Furga, Giulio; Brügger, Britta; Bartenschlager, Ralf
title: Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation
date: 2021-05-10
journal: bioRxiv
DOI: 10.1101/2021.05.10.443480
sha: 70d1e45a23bd874a6c162bc929ad29968853bcbc
doc_id: 1039002
cord_uid: dox32hb7

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses. One Sentence Summary Phosphatidic acid is important for the formation of double membrane vesicles, serving as replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-targeting strategy to treat SARS-CoV-2 infection.

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically 43

and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe 44 acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically 45 analogous to DMVs formed during autophagy, and although the proteins required for DMV 46

formation are extensively studied, the lipids driving their biogenesis are largely unknown. 47

Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate 48 acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral 49

replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that 50

AGPATs are recruited to and critically contribute to HCV replication and DMV formation. 51 AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of 52 autophagosome-like structures. By using correlative light and electron microscopy, we 53

observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In 54 addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent 55

with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from 56

AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and 57 diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired 58

HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. 59

These data identify PA as an important lipid used for replication organelle formation by HCV 60

and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. 61

chronic liver disease and COVID-19, respectively. In addition, our data argue that host-62

targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication 63 of these viruses. 64 65

Phosphatidic acid is important for the formation of double membrane vesicles, serving as 67 replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-68 targeting strategy to treat SARS-CoV-2 infection. 69 70 71

Chronic hepatitis C and COVID-19 are major medical problems. Both diseases are 73 caused by viral infections inflicting a large number of people and having led to millions of 74 deaths 1, 2 . Chronic hepatitis C is caused by persistent infection with the hepatitis C virus 75

(HCV), while COVID-19 is due to acute infection with the severe acute respiratory syndrome 76

coronavirus-2 (SARS-CoV-2). Both viruses are biologically very distinct e.g. by having a 77 very narrow tropism and a predominantly persistent course of infection in the case of HCV, 78

contrasting the rather broad tropism and acute self-limiting course of infection in the case of 79 SARS-CoV-2. This biological distinction is reflected by their phylogenetic distance with 80 HCV belonging to the Flaviviridae and SARS-CoV-2 being a member of the Coronaviridae 81 virus family 3 . In spite of these differences, both viruses possess a single strand RNA genome 82 of positive polarity that is replicated in membranous vesicles in the cytoplasm of infected 83 cells 4, 5 . These vesicles are induced by viral proteins, in concert with cellular factors, and 84

composed of two membrane bilayers, thus corresponding to double-membrane vesicles 85

(DMVs). These DMVs accumulate in infected cells and can be regarded as viral replication 86

organelle. Viral DMVs have morphological similarity to autophagosomes 6, 7 , but while 87 autophagy-induced DMVs serve to engulf cellular content and damaged organelles for 88 subsequent degradation, viral DMVs create a conducive and protective environment for 89 productive viral RNA replication. In the case of HCV and SARS-CoV-2, DMVs are derived 90 from the ER 8, 9, 10 and can be induced by the nonstructural proteins (NS)3, 4A, 4B, 5A and 91 5B in the case of HCV 7 and the viral proteins nsp3-4 in the case of MERS-CoV and SARS-92

CoV 11, 12 , alongside with co-opted host cell proteins and lipids. Here, we set-out to search for 93 common host cell factors exploited by the phylogenetically distant HCV and SARS-CoV-2 to 94 build up their cytoplasmic replication organelle. 95

Using HCV as a model to study DMV biogenesis, we purified DMVs under native 96

conditions and determined their molecular composition by proteomic profiling (Fig. 1A and  97 B). To this end we used human hepatoma cells (Huh7) containing a self-replicating HCV 98

replicon RNA (designated sg4B HA 31R; 13 ) in which NS4B was HA-tagged ( fig. S1A ). This 99 RNA replicates autonomously and induces an extensive array of DMVs that can be isolated 100 by HA-affinity purification 13 . Mass spectrometry-based proteomics analysis identified a total 101

of 1487 proteins significantly enriched in the NS4B-HA sample relative to the untagged 102 technical negative control (using SAINT average P-values >0.95) (data S1). Label free 103 quantitation (LFQ) revealed a major overlap of proteins (1542) between the NS4B-HA 104 complex and HCV-naïve ER membranes purified in parallel from Huh7 cells stably 105 expressing HA-tagged Calnexin (CNX-HA) ( Fig. 1B and fig. S1B ). Of note, 309 proteins 106

were significantly enriched in the NS4B-HA sample relative to the ER control with an over-107

representation of proteins involved in RNA metabolism, intracellular vesicle organization and 108 transport as well as endomembrane organization ( fig. S2 ). Given our interest in identifying 109

proteins of relevance for DMV formation, we selected 139 candidates with a bias for proteins 110 involved in vesicle transport and biogenesis as well as lipid metabolism. These candidates 111

were validated with respect to their role in HCV replication by using RNA interference-based 112 screening ( Fig. 1C and data S2). In this way we could validate 38 hits as HCV dependency 113

factors. Amongst identified hits were acylglycerolphosphate acyltransferase (AGPAT) 1 and 114 2, two enzymes that catalyze the de novo formation of phosphatidic acid (PA), a precursor to 115 di-and triacylglycerols as well as all glycerophospholipids 14, 15 . In addition, PA is involved 116

in signaling and protein recruitment to membranes and, owing to its small and highly charged 117 head group, promotes membrane curvature 16, 17, 18 . Since these properties might be involved 118

in DMV formation, we focused our subsequent analysis on AGPATs.

AGPATs play crucial roles in lipid homeostasis, because enzyme-inactivating mutations 120

in AGPAT2 are linked to congenital generalized lipodystrophy and defects in PA metabolism 121 as well as autophagy are associated with neurological disorders and chronic obstructive 122 pulmonary disease 18, 19 . Moreover, severe lipodystrophy as well as extreme insulin resistance 123 and hepatic steatosis have been observed in AGPAT2 -/mice 14 . To date, 11 AGPATs have 124 been identified in mammalian cells. AGPAT1 to 5 preferentially utilize lysophosphatidic acid 125

(LPA) as an acyl donor while AGPAT6 to 11 preferentially utilize alternative 126 lysophospholipid substrates or have a preference for glycerol-3-phosphate. Thus, only 127 AGPAT1 to 5 function as true LPA acyltransferases 14 . To establish which AGPAT family 128 members are found in NS4B-associated membranes, FLAG-tagged versions of each of the 5 129

AGPATs were transiently expressed in cells containing the HCV replicon sg4B HA 31R ( fig.  130 S3A). Pull-down of NS4B-HA revealed association with AGPAT1 and 2, and to a lesser 131 extent with AGPAT3, but not with AGPAT4 and 5. Additionally, endogenous AGPAT1 and 132 2 were detected in NS4B-HA containing membranes isolated from replicon-containing cells 133 (Fig. 1D ), whereas AGPAT 3 was not enriched. Moreover, in HCV infected cells AGPAT1 134 and 2 were recruited to NS4B-containing sites that most likely correspond to sites of DMV 135 accumulation 13 (Fig. 1E ).

To validate the role of AGPAT1 and 2 in HCV replication, we created knock-out cells 137

using CRISPR/Cas9. Although we observed reduced cell growth of stable double knock-out 138 HCV reporter virus and viral replication was determined by using luciferase assay. While 145 single KO suppressed HCV replication by ~50-70%, a reduction by ~90% was observed in 146 DKO cells (Fig. 1F ). Even stronger replication suppression was observed with a subgenomic 147 replicon ( fig. S4A ), confirming that AGPAT depletion affected viral RNA replication and not 148

virus entry or assembly. Of note, replication was completely restored by stable expression of 149 AGPAT1 and 2 in DKO cells, which was not the case with either or both enzymatically 150

inactive mutants (Fig. 1G ). In contrast, replication of Dengue virus (DENV) and Zika virus 151

(ZIKV), also belonging to the Flaviviridae family, but inducing morphologically different 152 membrane alterations, i.e. ER membrane invaginations 4 , was not affected as determined by 153 plaque assay or with a reporter virus ( Fig. 1H and fig. S4B , respectively). These results 154

suggest that enzymatically active AGPAT1 and 2 are required for HCV replication with both 155

AGPATs having partially redundant functions. 156

Next, we determined the impact of AGPAT KO on HCV-induced DMV formation. Since 157 AGPAT1/2 DKO reduces RNA replication, we employed a replication-independent system in 158

which DMV production is induced by the sole expression of an HCV NS3-5B polyprotein 159 fragment that undergoes self-cleavage to produce functional NS3, 4A, 4B, 5A and 5B 8, 22 160 ( Fig. 2A) . To determine the replicase subcellular location by fluorescence microscopy, NS5A 161 was fluorescently tagged with EGFP. This tagging has no effect on replicase functionality 8, 22 . 162

While expression of this polyprotein induced a high number of DMVs in control cells, DMV 163

abundance was dramatically reduced in AGPAT1/2 DKO cells (Fig. 2, A and B) , although 164

amounts of viral proteins were comparable in control and DKO cell pools (Fig. 2C ). 165

Moreover, DMVs had a smaller diameter in AGPAT2 KO cells ( fig. S4C ). These results 166

argue for a pivotal role of AGPATs in HCV DMV biogenesis. 167

Given that AGPAT1 and 2 are important for DMV formation and their enzymatic 168 activity is required for HCV replication, we next focused on their reaction product, i.e. the 169 lipid PA. To quantify the amount of PA associated with HCV-induced DMVs and compare it 170

to ER membranes, we determined the lipidome of highly purified DMVs isolated from cells 171

containing the sg4B HA 31R replicon (Fig. 2D ). Consistent with earlier results, these 172 membranes contained elevated amounts of cholesterol and sphingolipids, which served as 173 positive controls, relative to ER membranes purified in parallel 13, 23 . Of note, PA abundance 174

in DMVs also was increased in comparison to ER membranes, whereas the level of diacyl 175 phosphatidylcholine (aPC) and several other lipids was not affected ( Fig. 2D ; for further 176 lipids see data S3). 177

To confirm these findings in single cells, we used two alternative methods to detect PA 178 by fluorescence microscopy. First, we generated a recombinant protein composed of GST 179

that was fused to the PA binding domain (PABD) derived from yeast Spo20p ( fig. S5A and  180 B). As a specificity control we employed the analogous sensor protein containing a mutation 181

in the PABD that abolishes PA binding, and GST alone 24 197 Since these data suggest an important role of AGPAT1 and 2-dependent PA 198 enrichment on HCV-induced DMVs, we hypothesized that other pathways contributing to PA 199 generation in cells might also play a role in HCV replication. Apart from AGPATs, one other 200 route for PA synthesis is through hydrolysis of phosphatidylcholine (PC) by phospholipase 201 D1 (PLD1) and D2 (PLD2) enzymes (Fig. 2F , top panel) 17, 27 . To test the role of PLD1/2 202 enzymes in HCV replication, we employed a pharmacological approach using 3 different 203 PLD1/2 inhibitors. Treatment with PLD2 inhibitor ML298 caused replication inhibition at a 204 concentration that did not significantly reduce cell viability (~25 µM; Fig. 2F , bottom panel), 205

whereas for the other drugs the reduction in HCV replication correlated with cytotoxicity (not 206

shown). In summary, these results suggest that PA generated via AGPAT1/2, and possibly by 207

alternative PA synthesis pathway, contributes to HCV replication by supporting the formation 208

of DMVs, which is the site of viral RNA amplification. 209

Virus-induced DMVs are morphologically analogous to autophagosomes generated 210 during autophagy 7 ; therefore, we tested if PA would be recruited to and is required for suggesting that PA generated on the ATG16L1-positive autophagosome precursor membrane 230 contributes to autophagosome formation 30 . Of note, a third pathway for PA production via 231 phosphorylation of diacylglycerol (DAG) by diacylglycerol kinase (DAGK) 27 , did not 232 contribute to PA accumulation or increase in LC3 puncta during nonselective autophagy ( fig.  233 S7). 234

Having found that AGPAT1 and 2, and their reaction product PA, are involved in DMV 235

formation induced upon HCV infection and in, morphologically similar, DMVs generated 236 during autophagy, we hypothesized that AGPATs and PA might also be involved in the 237 biogenesis of replication organelles of other unrelated RNA viruses, e.g., coronaviruses, 238

which also utilize DMVs as viral replication sites 9, 10 . Hence, we investigated the role of 239

AGPATs in the DMV biogenesis of SARS-CoV-2, the causative agent of the ongoing 240 COVID-19 pandemic. In the first set of experiments, we studied the recruitment of AGPATs 241

to SARS-CoV-2 induced DMVs. In the case of MERS-CoV and SARS-CoV, formation of 242

DMVs with structural resemblance to those observed in infected cells can be induced by the 243 sole expression of viral nonstructural protein (nsp)3-4, which is an ~270 kilodalton large 244 polyprotein fragment undergoing self-cleavage 12 . Building on these results we first 245 determined whether the same applies to SARS-CoV-2. Huh7-derived cells stably expressing 246 T7 RNA polymerase were transiently transfected with a T7 promoter driven SARS-CoV-2 247

HA-nsp3-4-V5 expression construct or the empty vector ( fig S8A) . Using 248

immunofluoresence with an HA-specific antibody in many cells we observed clusters of HA-249

nsp3 ( to ~300 nm, respectively) ( fig S8E) . These results show that the sole expression of SARS-258

CoV-2 nsp3-4 is sufficient to induce DMVs with structural similarity to those generated in 259 infected cells. 260

Next, we employed this expression-based system to determine AGPAT function in 261 SARS-CoV-2 nsp3-4 induced DMV formation. Huh7-derived cells expressing GFP-tagged 262 AGPAT1 or 2 were transiently transfected with the SARS-CoV-2 HA-nsp3-4-V5 encoding 263 plasmid or the empty vector and colocalization of AGPATs with HA-nsp3 was determined by 264

immunofluorescence microscopy. While in empty vector-transfected cells AGPAT2 and 1 265

were homogeneously distributed throughout the ER ( Fig. 3A and fig. S9A , respectively), we 266 observed a strong relocalization of AGPATs in HA-nsp3-4-V5 expressing cells with 267

AGPATs forming puncta that colocalized with HA-nsp3 (Fig. 3, A and B; fig. S9A ). Of note, 268

the relocalization of AGPATs induced by HA-nsp3-4-V5 was not the result of the massive 269 ER alterations occurring in SARS-CoV-2 infected cells, since the subcellular distribution of 270 other ER resident proteins, such as protein disulfide-isomerase (PDI) and calnexin remained 271 unaffected compared to the large puncta observed with AGPATs (Fig. 3C ). Since SARS-272

CoV-2 replication organelles are comprised of DMVs, convoluted membranes and zippered 273 ER 31 , we next investigated the membrane structures at the sites of AGPAT colocalization 274

with HA-nsp3-4-V5. Using correlative light electron microscopy, we found that relocalized 275

AGPAT puncta perfectly correlated with extensive networks of SARS-CoV-2 HA-nsp3-4-V5 276

induced DMVs (Fig. 3D ). Overall, the data shown here suggest that similar to HCV, 277

AGPATs are relocalized to SARS-CoV-2 nsp3-4 induced DMVs, the likely sites of viral 278 RNA replication 32 . 279

Next, we tested the effect of AGPAT1/2 depletion on SARS-CoV-2 infection and 280

replication. To this end we used DKO Huh7-Lunet/T7 cells that were employed for the 281 imaging analyses described so far and stably introduced the SARS-CoV-2 receptor gene 282

ACE2. Viral replication was measured by using an image-based assay that quantifies the 283 number of cells containing detectable amounts of the nucleocapsid (N) protein ( fig. S9B ). 284

Using this approach, we observed significant reduction of SARS-CoV-2 positive cells in both 285 single and double AGPAT knockout cells (Fig. 3E) . Consistently, RT-qPCR revealed similar 286

reduction of viral replication in single and double KO cells (Fig. 3E, lower right panel) . To 287 determine if reduced SARS-CoV-2 replication in AGPAT1/2 KO cells might correlate with 288 altered DMV formation, we transiently expressed SARS-CoV-2 HA-nsp3-4-V5 in control, 289

single and double KO cells. The absence of AGPAT 1/2 did not significantly affect the 290 abundance of cleaved viral proteins HA-nsp3 and nsp4-V5 ( fig. S8C ). EM analysis of control 291 cells revealed HA-nsp3-4-V5 induced membrane alterations, consistent with an earlier report 292

for MERS-CoV and SARS-CoV 12 (Fig. 3, F and G) . This included zippered ER and DMVs 293

with an average diameter of 145 nm. In contrast to HCV, the number of nsp3-4 induced 294

DMVs did not decrease in AGPAT single and double KO cells (Fig. 3G , left two panels). 295

However, in both cell pools we observed marked accumulations of multi-membrane vesicles 296

(MMVs), indicating the formation of aberrant membrane structures (Fig. 3, F and G) . 297

To test whether similar to AGPAT1/2 relocalization to nsp3-4 induced DMVs, PA is also 298 enriched at those sites we used the GFP-tagged PA sensor derived from Raf1. In Huh7-299 derived cells expressing SARS-CoV-2 HA-nsp3-4-V5, the functional version of the sensor 300

(GFP-PABD-Raf1-WT) strongly colocalized with HA-nsp3 in distinct puncta, whereas no 301 such puncta were found with the mutant PABD-Raf1, confirming specificity of PA sensor 302 recruitment to HA-nsp3-containing sites (Fig. 4, A and B) . 303

Although in comparison to HCV, AGPAT1/2 DKO had lower impact on SARS-CoV-2 304

replication (compare Fig. 1F with Fig. 3E ), and caused a morphologically distinct phenotype 305 of nsp3-4 induced DMVs ( Fig. 2A and 3F, respectively) , AGPATs, and most likely PA, still 306 accumulated at sites of SARS-CoV-2 DMV clusters (Fig. 4, A and B ). This indicates that PA 307 synthesis pathways other than via AGPAT1/2, might contribute to SARS-CoV-2 replication 308 and DMV formation. By means of pharmacological inhibitors of enzymes that convert LPA, 309

PC and DAG to PA ( fig. S7A ), we measured the dose-dependent effect of these drugs on 310

SARS-CoV-2 replication. All inhibitors reduced SARS-CoV-2 replication in Calu-3 cells and 311

in A549 cells stably expressing ACE2, two commonly used cell models for this virus, at non-312 cytotoxic concentrations, although in the case of the general AGPAT inhibitor CI976 313 selectivity was rather low ( Fig. 4C and fig. S10A , respectively). Of note, combining the 314 inhibitors at concentrations close to or below their IC50 values caused much stronger 315 reduction of virus replication with no or minimal effect on cell viability, indicating that 316 SARS-CoV-2 can utilize PA produced by alternative PA synthesis pathways (fig. S10, A and 317 B). We then measured the effect of these drugs on PA accumulation at HA-nsp3 containing 318 puncta in HA-nsp3-4-V5 expressing cells and found that all inhibitors reduced PA levels at 319 these sites (Fig. 4D ). This reduction was not the result of altered HA-nsp3-4-V5 expression 320 level or self-cleavage, which were unaffected in inhibitor-treated cells ( fig. S10C ). Next, we 321 determined if reduced PA levels caused by these inhibitors also affect SARS-CoV-2 nsp3-4 322 induced DMV formation. In cells treated with AGPAT, PLD1, and DAGK inhibitors DMV 323 diameters were significantly reduced (Fig. 4 , E and F). Moreover, PLD2 inhibition promoted 324 the formation of MMVs and larger DMVs, similar to what we found in AGPAT single and 325

double KO cells (Fig. 3F ). Taken together, our data suggest that PA enrichment is important 326

for proper SARS-CoV-2 DMV formation and viral replication. 327

Here, we show that PA produced by AGPAT1 and 2 is important for the replication of 328 evolutionary distant positive-strand RNA viruses, HCV and SARS-CoV-2 that amplify their 329 genome in association with DMVs. The remarkable dependence on a common host lipid for 330 the DMV biogenesis in these two viruses that differ profoundly in the diseases they cause and 331

in their biological properties, indicates a striking similarity in the biogenesis of these 332 organelles. Conversely, for viruses replicating their RNA genome in ER-derived membrane 333

invaginations such as the flaviviruses DENV and ZIKV, this lipid pathway appears to be 334 dispensable 4, 33 . Of note, PA production through AGPAT1 and 2 is also involved in the 335 formation of autophagosome-like DMVs, arguing for some similarity between cellular and 336 viral DMV formation and lipid composition. Additionally, alternative routes of PA 337 biosynthesis contribute to HCV and SARS-CoV-2 replication and DMV generation.

At least three possibilities can be envisioned how PA promotes DMV formation in 339 viral replication and in the context of autophagy. First, the presence of lipids with cone or 340 inverted cone shape in membranes contributes to membrane bending by generating negative 341 or positive membrane curvature, respectively 16 . While LPA has a large polar head group to 342 fatty acid tail ratio, giving rise to an inverse-cone shape and resulting in positive membrane 343 curvature, the additional fatty acid tail present in PA inverses the head-to-tail ratio. Hence PA 344

displays an overall cone shape, which contributes to negative membrane curvature. 

Authors declare no competing interests. 534 535

All data is available in the main text or the supplementary materials. 537 538

Figures S1-S10 541

Tables S1-S5 542

References ( containing the subgenomic replicon sg4B HA 31R (NS4B-HA) and Huh7 cells stably 602

overexpressing HA-tagged Calnexin (CNX-HA) and control Huh7 cells were prepared as 603 described in supplementary methods and used for HA-affinity purification under native 604

conditions. An aliquot of the sample was analyzed by electron microscopy (top panels) 605

whereas the majority was subjected to lipidome analysis by using mass spectrometry. Values 606 obtained for the NS4B-HA sample were normalized to those obtained for the CNX-HA 607

sample that was set to one. The complete list of analyzed lipids is summarized in data S3. (E) 608

PA accumulation at NS5A containing structures. Huh7-Lunet/T7 cells were transfected with 609 a construct analogous to the one in panel A, but containing a mCherry insertion in lieu of 610 GFP, along with an EGFP-tagged wildtype ( Huh7-derived cells transiently expressing AGPAT2-GFP were transfected with a SARS-636

CoV-2 HA-nsp3-4-V5 expression construct or the empty vector. After 48h, cells were stained 637

Maximum intensity 638 projections are shown. Enrichment score indicates the likelihood of cells showing a punctate 639 or diffuse staining pattern. (B) Clustering of AGPAT2-GFP in SARS-CoV-2 HA-nsp3-4-V5 640 expressing cells. Huh7-Lunet/T7 cells were co-transfected with

CoV-2 HA-nsp3-4-V5 or the empty vector. Twenty-four hours later, cells were fixed and 642 ~1000 cells per condition were separated into two morphotypes (diffuse or punctate)

Significance was calculated using an 644 unpaired t-test. *, p<0.05. (C) AGPAT clustering occurs independent of ER remodeling 645 induced by nsp3-4. Huh7-Lunet cells expressing AGPAT2-GFP and HA-nsp3-4-V5 were 646 stained for the ER markers protein disulfide isomerase (PDI) and calnexin and analyzed by 647 confocal microscopy

Significance was calculated using ordinary one-way ANOVA. *, p<0.05. (F) Aberrant 659 SARS-CoV-2 DMVs in AGPAT1/2 DKO cells. Huh7-Lunet cells with single (SKO) or 660 double knock-out (DKO) and stably expressing T7 polymerase were transfected with a 661 plasmid encoding SARS-CoV-2 HA-nsp3-4-V5 and fluorescent neon-green. Twenty-four 662 hours later, cells were fixed and NeonGreen positive cells were recorded and examined by 663 EM. HA-nsp3-4-V5 induced DMVs and multi-membrane vesicles (MMVs) were quantified. 664 Shown are the number and diameter of DMVs and MMVs in these cells as observed from at 665 least 8 cell profiles per condition

PA accumulation at SARS-CoV-2 DMVs and role of alternative PA synthesis 675 pathways for SARS-CoV-2 replication and DMV formation. (A) PA enrichment at 676 SARS-CoV-2 nsp3-containing structures. Huh7-Lunet cells expressing the wildtype or 677 mutant form of the PA sensor were transfected with the plasmid encoding HA-nsp3-4-V5

HA-specific antibody and HA-nsp3 and 679 GFP-PABD were visualized by confocal microscopy. Maximum intensity projections are 680 shown. (B) Using CellProfiler Analyst, a semi-supervised machine learning classifier was 681 trained to differentiate between punctate and diffuse signals of the GFP-PABD sensor (top 682 panel). A normalized enrichment score which indicates the probability of cells showing 683 punctate GFP-PABD localization to nsp3 fluorescent signal across the whole cell population 684 is shown in the graph on the bottom panel

C) Alternative pathways for PA generation are important for SARS-CoV-2 686 replication. Calu-3 cells were infected with SARS-CoV-2 (MOI=5) in the presence of 687

PLD1/2, or DAGK inhibitors. Cells were fixed 24 h post infection, stained with 688 nucleocapsid-specific antibody and percentage of infected cells was quantified using 689

Huh7-Lunet cells were transfected with 692 SARS-CoV-2 HA-nsp3-4-V5 and GFP-PABD-Raf1 encoding plasmids, followed by addition 693 of a given inhibitor 4h after transfection. Twenty-four hours later, cells were fixed and HA-694 nsp3 was detected with an HA-specific antibody. GFP-PABD and HA-nsp3 were visualized 695 by confocal microscopy. A semi-automated machine learning based classifier was trained to 696 separate HA-nsp3/PABD double-positive structures from HA-nsp3 single positive structures. 697 Enrichment score for HA-nsp3/PABD double-positive structures showing the up or 698 downregulation of double positive cells in different samples is plotted and statistical 699 significance was calculated using ordinary one-way ANOVA

Lunet/T7 cells were transfected with the plasmid encoding HA-nsp3-4-V5 and fluorescent 702

Twenty-four hours later, 703 cells were fixed, NeonGreen positive cells were recorded and examined by EM. 704 Representative images are shown for each condition. (F) Number and morphology of DMVs 705 were determined for at least 7 cell profiles per condition. DMV diameters are plotted and 706 statistical significance was calculated using ordinary one-way ANOVA