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SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation

Hye Jin Shin Korea Research Institute of Chemical Technology Keun Bon Ku Korea Research Institute of Chemical Technology Gun Young Yoon Korea Research Institute of Chemical Technology Hyun-Woo Moon Korea Research Institute of Chemical Technology Chonsaeng Kim Korea Research Institute of Chemical Technology Bum-Tae Kim Korea Research Institute of Chemical Technology Jong-Won Oh Yonsei University Aleem Siddiqui UC San Diego Seong-Jun Kim (  sekim@krict.re.kr ) Korea Research Institute of Chemical Technology https://orcid.org/0000-0001-8504-2872


Introduction 39
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat leading to 40 serious respiratory illness and death since the first case was reported as a fatal pathogen in 2019 1,2 . 41 SARS-CoV-2 phylogenetically closely related to SARS-CoV causing an epidemic in 2003 3,4 is an 42 enveloped, single-stranded positive-sense RNA virus belonging to the family Coronaviridae 5 . SARS-43 CoV-2 enters into the host cells via host factors angiotensin-converting enzyme carboxypetidase 44 (ACE2) and transmembrane serine protease 2 (TMPRSS2) and utilizes own RNA-dependent RNA 45 polymerase (RdRp) for the replication of viral genome in the cytoplasm 6-8 . In particular, when SARS-46 CoV-2 infects cells by ACE2-mediated endocytosis, SARS-CoV-2 nucleocapsid protein binding to the 47 packaging signals in the virus RNA (SARS-CoV-2 RNA-nucleocapsid cluster) enters into the cells 9,10 48 unlike other RNA viruses such as hepatitis C virus (HCV) and Zika virus (ZIKV) and so on 29,30 . 49 Viruses manipulate various intracellular events such as endoplasmic reticulum (ER) stress, 50 oxidative stress, and Ca 2+ flux between ER and mitochondria that affects viral propagation and 51 inflammation 11-14 . Particularly, virus infection induces mitochondrial Ca 2+ overload thereby resulting 52 in collapse of mitochondria membrane potential (ΔΨm) which is the key step in modulating 53 mitochondrial dynamics for the maintenance of cellular homeostasis 15,16 . Recent evidences showed 54 that virus infection induces abnormal mitochondrial dynamics and perturbs mitochondrial 55 bioenergetics that are critical for viral propagation and antiviral innate immune response 17,18 . 56 The regulation of homoeostasis in host cells is important for the replication of coronaviruses 19 . 57 The pro-survival function of epidermal growth factor receptors (EGFR) is involved in many viral 58 infections. The activation of EGFR is not only required for viral entry to the plasma membrane but 59 also for mitochondrial biogenesis and bioenergetics for viral replication [20][21][22][23][24][25] . Recent reports showed 60 that beyond a significant contribution of EGFR signaling required for sustaining cell survival and 61 proliferation, EGFR migration to subcellular organelles such as ER and mitochondria following EGFR 62 into the cells when SARS-CoV-2 infects host cells by ACE2-mediated endocytosis 9,10 . Hence, we 111 sought to analyse ΔΨm in the presence of SARS-CoV-2 RNA and nucleocapsid protein. As shown in 112 Fig. 1c, we first analysed that the expression of SARS-CoV-2 nucleocapsid alone does not significantly 113 affect ΔΨm (Fig. 1c, left graph). Next, we found that the transfection of SARS-CoV-2 RNA in cells 114 causes loss in ΔΨm (right graph), whereas the transfection of SARS-CoV-2 RNA in cells expressing 115 nucleocapsid protein induces dramatic increases in ΔΨm (Fig. 1c, right graph). poly(I:C) was used for 116 the control for ΔΨm loss 32 . poly(I:C) did not induce ΔΨm increase in the presence and absence of 117 SARS-CoV-2 nucleocapsid protein, suggesting that the elaborate structure or formation of SARS-CoV-118 2 ssRNA and nucleocapsid protein might affect ΔΨm increase. The accompanying schematic diagram 119 represents that the expression of SARS-CoV-2 RNA and nucleocapsid together causes ΔΨm increase 120 ( Fig. 1c). 121 Next, we examined a mitochondrial shape on confocal microscope, as change in ΔΨm usually 122 causes alteration in mitochondrial morphology and induces abnormal mitochondrial function. As 123 shown in Fig. 1d, we observed that aberrant elongated and swollen mitochondrial shape is sustained 124 in SARS-CoV-2-infected cells. The accompanying graph presents the quantification of mitochondrial 125 length in SARS-CoV-2-infected cells and uninfected cells shown in confocal microscope. Lack of 126 mitofusin (MFN) creates mitochondrial fusion deficiency and causes severe mitochondrial 127 fragmentation 33 . Hence, we examined the stability of MFN in SARS-CoV-2-infected cells. As shown 128 in Fig. 1e, we found that MFN1/2 is increased and sustained in SARS-CoV-2-infected cells, 129 demonstrating that MFN acts as a modulator for sustaining abnormal mitochondrial elongation in 130 SARS-CoV-2-infected cells. We also examined mitochondrial biogenesis through analysis of 131 mitochondrial DNA (mtDNA) level in SARS-CoV-2-infected cells. Analysis of mitochondrial NADH 132 dehydrogenase 2 (ND2) and cytochrome c oxidase 2 (COX2) reveals that the abnormal increase in 133 mitochondrial mass is maintained during SARS-CoV-2 replication (Fig. 1f). 134 Most of the ATP is mainly generated in mitochondria by the oxidative phosphorylation (OXPHOS) 135 process 34 . Thus, we subsequently examined a change in the expression levels of mitochondrial 136 OXPHOS complex that might be associated with abundant ATP production on SARS-CoV-2-induced 137 abnormal mitochondrial fusion. As a result, we found that the expression levels of mitochondrial 138 OXPHOS complex I, II, III, IV, and V enzymes are entirely increased in SARS-CoV-2-infected cells 139 (Fig. 1g). Further study by RNAseq and subsequent analysis demonstrated that most of the genes 140 involved in OXPHOS process were upregulated in SARS-CoV-2 (Fig. 1h). Taken together, these 141 results indicate that SARS-CoV-2 induces increase in ΔΨm, mitochondrial biogenesis, and OXPHOS 142 process, thereby abnormally promoting ATP production. 143 144

SARS-CoV-2 activates EGFR signal cascade and subsequently induces mitochondrial EGFR 145 translocation 146
Next, we observed the prolonged survival and proliferation of human cells infected with SARS-147 CoV-2 without a distinct viral cytopathogenic effect while we analyse SARS-CoV-2 infectivity in 148 various human cells Extended Data Fig.3). Thus, we sought to 149 analyse SARS-CoV-2-induced activation in EGFR signal cascade, as contribution of EGFR signaling 150 is critical for sustaining cell survival and increased cell proliferation 35,36 . As shown in Fig. 2c, SARS-151 CoV-2 promoted EGFR gene expression at the transcriptional levels at an early time point of SARS-152 CoV-2 infection. Subsequent Western blot analysis also revealed that SARS-CoV-2 results in activation 153 of EGFR-mediated signal cascade as evidenced by increase in the expression levels of phosphorylated 154 EGFR and phosphorylated Akt (Fig. 2d). Together, these results indicate that SARS-CoV-2 promotes 155

EGFR-mediated cell survival and proliferation of infected cells. 156
Beyond a significant contribution of EGFR signal cascade required for sustaining cell survival and 157 proliferation, EGFR migration to subcellular organelles following EGFR activation has been 158 reported [36][37][38] . Particularly, mitochondrially localized EGFR contributes to cell survival and regulation 159 of mitochondrial function 36 . Hence, we further examined if SARS-CoV-2 induces EGFR translocation 160 to mitochondria. We isolated the mitochondrial fraction from SARS-CoV-2-infected cells and analysed 161 SARS-CoV-2-induced mitochondrial EGFP translocation by Western blotting with antibodies specific 162 to phospho-EGFR and EGFR, respectively. As shown in Fig. 2e, SARS-CoV-2 elevated mitochondrial 163 translocation of EGFR and its accumulation compared to those in uninfected cells. A quantitative 164 analysis reveals the difference in relative EGFR accumulation on mitochondrial fraction of SARS-165 CoV-2-infected cells (Fig. 2e). Further analysis by confocal microscopy also identified EGFR 166 accumulation on mitochondria of SARS-CoV-2-infected cells (Fig. 2f). Here, we note that SARS-CoV-167 2 nucleocapsid is localized on mitochondria as evidenced by Western blot analysis and confocal 168 microscopy using antibody specific to SARS-CoV-2 nucleocapsid antigen . 169 To strengthen this observation in vivo animal model, we first investigated SARS-CoV-2 infectivity 170 using various age groups of wild-type C57BL/6 mouse to establish wild-type mouse model that is 171 proper for studying SARS-CoV-2 pathogenesis, as we considered that wild-type mouse model is more 172 appropriate for studying SARS-CoV-2 pathogenesis than hACE2 transgenic mouse model which 173 induces an extreme infection of SARS-CoV-2 via overexpression of hACE2 gene. As a result, we 174 found that 15-month aged group of wild-type C57BL/6 mouse is susceptible to SARS-CoV-2 infection 175 (Extended Fig. 4). SARS-CoV-2-infected wild-type 15-month aged mice revealed changes in body 176 weight, survival rate, and viral infectivity in the lung tissue which is very similar with those shown in 177 Golden hamster and Syrian hamster (Extended Fig. 4) 39,40 . Subsequently, we isolated mitochondrial 178 fraction from the lung tissue of wild-type 15-month aged mouse infected with SARS-CoV-2 and 179 analysed mitochondrial EGFR accumulation by Western blotting with anti-EGFR antibody. As shown 180 in Fig. 2g, SARS-CoV-2 infection resulted in enhanced mitochondrial EGFR accumulation in the lung 181 tissue compared to those of uninfected wild-type 15-month aged mouse. A quantitative analysis shows 182 the difference in relative EGFR accumulation on mitochondrial fraction of lung tissue of SARS-CoV-183 2-infected mouse (Fig. 2e). Taken together, these results indicate that SARS-CoV-2 activates EGFR 184 signal cascade followed by mitochondrial EGFR accumulation during viral infection. 185 186 EGFR inhibitors reveals the potent antiviral activity against SARS-CoV-2 187 Next, we wondered if robust SARS-CoV-2 propagation is suppressed by inhibiting of SARS-CoV-188 2-activated EGFR signal cascade. Hence, we evaluated antiviral activity of FDA-approved 12 EGFR 189 inhibitors including gefitinib, olmutinib, erlotinib, lapatinib, bosutinib, cabozantinib, icotinib, 190 vandetanib, brigatinib, afatinib, dacomitinib, and neratinib in SARS-CoV-2-infected cells (Fig. 3a). 191 As shown in Fig. 3b, treatment of SARS-CoV-2-infected cells with EGFR inhibitors caused drastic 192 reduction in the level of extracellular SARS-CoV-2 RNA. Western blot analysis also exhibited that 193 EGFR inhibitors result in the reduction in the expression level of SARS-CoV-2 nucleocapsid protein 194 (Fig. 3c). Among EGFR inhibitors we tested, afatinib, dacomitinib, and neratinib induced a striking 195 cytotoxicity in SARS-CoV-2-infected cells (Table 2). Together, these results suggest that  approved EGFR inhibitors can be utilized as an antiviral therapeutic agent against SARS-

EGFR inhibitor restores SARS-CoV-2-induced abnormal mitochondrial bioenergetics 200
To substantiate the action of EGFR inhibitors on inhibition of SARS-CoV-2 propagation, we 201 further examined the effect of EGFR inhibitors in SARS-CoV-2-induced mitochondrial alteration. The 202 FDA-approved vandetanib revealed the highest antiviral efficacy against SARS-CoV-2 infection 203 among EGFR inhibitors we tested (Fig. 3b, c). Hence, we examined if vandetanib is capable of 204 restoring SARS-CoV-2-induced abnormal OXPHOS process and subsequent increase in ATP synthesis. 205 We first analysed that vandetanib reduces mitochondrial EGFR accumulation in SARS-CoV-2-infected 206 cells (Fig. 3d). Further, treatment of SARS-CoV-2-infected cells with vandetanib dramatically restored 207 SARS-CoV-2-induced increase in intracellular ATP level and the expression levels of mitochondrial 208 OXPHOS complex I, II, III, IV, and V enzymes (Fig. 3e, f). In addition, RNAseq and subsequent 209 analysis demonstrated that vandetanib restores SARS-CoV-2-induced abnormal upregulation in most 210 of genes involved in OXPHOS process (Fig. 3g). Taken together, these results indicate that SARS-211 CoV-2-induced activation of EGFR signal cascade followed by mitochondrial EGFR translocation 212 contributes to SARS-CoV-2-induced abnormal mitochondrial dynamics and bioenergetics supporting 213 robust SARS-CoV-2 propagation. 214

EGFR inhibitor vandetanib exhibits the potent anti-SARS-CoV-2 activity 216
To substantiate the antiviral effect of vandetanib on SARS-CoV-2 propagation, we first analysed 217 cell cytotoxicity of vandetanib. As shown in Fig. 4a, vandetanib did not significantly affect viability 218 of both uninfected and SARS-CoV-2-infected cells. Vandetanib also led to dose-dependent reduction 219 in SARS-CoV-2 nucleocapsid expression as assessed by Western blot analysis (Fig. 4b). To further 220 understand the action of anti-SARS-CoV-2 effect of vandetanib, we analysed the expression level of 221 intracellular SARS-CoV-2 RNA in infected cells under post-treatment or pre-treatment of vandetanib. 222 As shown in Fig. 4c and d, intracellular SARS-CoV-2 RNA level was drastically declined by post-223 treatment of vandetanib, but not pre-treatment as evidenced by real-time qRT-PCR data, suggesting 224 that vandetanib affects an intracellular replication process during SARS-CoV-2 propagation. 225 We further analysed the in vivo anti-SARS-CoV-2 efficacy of vandetanib using wild-type 15-226 month aged mice. Vandetanib is initially developed as an orally available agent 41 . Hence, we orally 227 administrated vandetanib to SARS-CoV-2-infected mice at dose of 25 mg/kg daily and terminated 228 SARS-CoV-2-infected mice at 4 days post-infection to analyse intracellular SARS-CoV-2 RNA level 229 in lung tissue of mice (Fig. 4e). As a results, we analysed that vandetanib strongly suppresses SARS-230 CoV-2 propagation as assessed by qRT-PCR analysis (Fig. 4f). The intramuscular administration of 231 vandetanib in SARS-CoV-2-infected mice did not reveal anti-SARS-CoV-2 efficacy (Fig. 4f). 232 A combination regimen targeting both virus and host might be more effective for curing . Thus, we additionally tested the anti-SARS-CoV-2 effect of vandetanib in combination with 234 remdesivir at a 100-fold lower concentration than the IC50. The advantage of this regimen with EGFR 235 inhibitors in combination with remdesivir is the drastic reduction in toxicity caused by the continuous 236 administration of remdesivir to patients with COVID-19 42,43 . As shown in Fig. 4g, treatment of 237 remdesivir in combination with vandetanib caused an additive reduction in intracellular SARS-CoV-2 238 RNA level, suggesting that vandetanib can be utilized in combination with remdesivir without toxicity. The anti-SARS-CoV-2 activity of vandetanib was evaluated by qRT-PCR analysis and SARS-CoV-2 243 foci reduction assay. As shown in Fig. 5a, treatment of SARS-CoV-2 variants-infected cells with 244 vandetanib caused drastic reduction in the expression levels of extracellular SARS-CoV-2 RNA. 245 Furthermore, vandetanib potently inhibited the infectivity of various SARS-CoV-2 strains in a dose-246 dependent manner (Fig. 5b). SARS-CoV-2 strains-induced foci formation was completely inhibited at 247 10 μM of vandetanib (Fig. 5b). Taken together, these results suggested that vandetanib is a very 248 efficient inhibitor against various SARS-CoV-2 strains. 249 250

Discussion 251
Although the current COVID-19 pandemic caused by SARS-CoV-2 infection is a severe threat 252 globally, our understanding of persistent SARS-CoV-2 infection and the pathogenesis of COVID-19 253 is still insufficient. In this study, we show that SARS-CoV-2 alters intracellular events and abnormally 254 modulates mitochondrial bioenergetics for robust virus propagation. We found that SARS-CoV-2 255 increases ΔΨm which leads to the mitochondrial elongation and swelling. SARS-CoV-2 RNA and 256 nucleocapsid complex promoted ΔΨm increase. SARS-CoV-2 infection promoted mitochondrial 257 biogenesis and activated mitochondrial OXPHOS process to generate abundant ATP. We also found 258 that SARS-CoV-2 induces EGFR-mediated cell survival signal cascade and subsequently promotes 259 mitochondrial EGFR accumulation which contributes to maintenance of abnormal mitochondrial 260 bioenergetics. Overall, our results suggest that the SARS-CoV-2-modulated abnormal mitochondrial 261 bioenergetics is physiologically relevant to the maintenance of cellular homoeostasis of SARS-CoV-262 2-infected cells and robust SARS-CoV-2 replication and propagation (Fig. 6). 263 The involvement of EGFR internalization and transport to cellular organelles provides a favourable 264 environment for virus replication [20][21][22][23][24]44 . We showed that SARS-CoV-2 stimulates EGFR-mediated cell 265 survival signal and mitochondrial EGFR accumulation for the maintenance of cell proliferation and 266

homeostasis. SARS-CoV-2 infection dramatically triggered the translocation of EGFR to mitochondria 267
as demonstrated by analyses of subcellular fractions and confocal microscpe. Subsequently, SARS-268 CoV-2 maintained the abnormally elevated mitochondrial bioenergetics via EGFR internalization and 269 accumulation on mitochondria, which affects viral replication and ATP production. In hepatitis C virus 270 (HCV), EGFR acts as a co-factor for entry and upregulates its internalization and transport to the 271 cellular organelles, and similar processes have been also reported for hepatitis B virus (HBV) and 272 gastroenteritis virus [20][21][22][23][24][25]45 . SARS-CoV infection also involved the overactivation of EGFR, which 273 leads to increased pulmonary fibrosis 46 . Of note, many respiratory viruses induce EGFR activation in 274 the airway epithelium 24,47 . Moreover, the aberrant expression and activation of EGFR have been 275 implicated in multiple diseases including cancer, diabetes, pulmonary fibrosis, and various lung, heart, 276 and renal diseases 35,48-51 . 277 Notably, vandetanib caused a remarkable inhibition of SARS-CoV-2 propagation in our study 278 against all clades of SARS-CoV-2. Vandetanib, an FDA-approved EGFR inhibitor, is an inexpensive 279 drug with low toxicity that has been used for the treatment of certain tumours of the thyroid gland 52,53 . 280

Several EGFR inhibitors have already been shown to block multiple steps of replication in influenza 281
virus 54 and dengue virus 55 . Therapeutic options in response to the various SARS-CoV2 strains are still 282 limited. In the context of the current COVID-19 pandemic, our study suggests that EGFR inhibitors 283 are a druggable regulator to reduce viral replication by modulation of mitochondrial bioenergetics and 284 EGFR signal cascade against SARS-CoV-2 variants. Prevention. SARS-CoV-2 variants used in this study were listed in Table 1  phenylhydrazon, 400nM) was added into one well and incubated at 37°C for 10 min prior to TMRE 333 addition. Following incubation, the stained cells were harvested and gently washed with assay buffer. 334 The fluorescence signal was measured at 549/575 nm of excitation/emission wavelengths using a 335 Synergy H1 multi-mode microplate reader (BioTek). Two technical and biological replicates were 336 performed for each sample. 337 338

Real-time qRT-PCR and qPCR 339
To analyse the expression levels of EGFR and SARS-CoV-2 viral genes, total RNA was extracted 340 from cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Extracellular viral RNA from 341 supernatants was isolated using a QIAamp Viral RNA Mini Kit (Qiagen). The cellular RNA level of 342 EGFR was quantified by real-time qRT-PCR using the One-Step SyBr Green RT-PCR Kit II (Takara). 343 The intracellular and extracellular SARS-CoV-2 RNA copy numbers were determined by real-time 344 qRT-PCR using the One Step PrimeScript III RT-PCR Kit (Takara). The following primer sets were 345 used for RT-PCR: EGFR forward, 5′-AGGCAGGAGTAACAAGCTCAC; EGFR reverse, 5′- The titer of infectious virus in the culture medium was determined by FFU assay. Vero E6 cells 393 were seeded in a 96 well plate. The infectious cell culture medium was used to infect the cells and, the 394 plates were incubated at 37 °C. After 8 h, cells were washed with PBS and fixed with 4% 395 paraformaldehyde and permeabilized with methanol. SCoV2 foci were developed using anti-SCoV2

RNAseq analysis and data processing 403
Raw reads were mapped to the human genome assembly hg38 using the aligner STAR v.2.4.0b 57 . 404 To measure gene expression level, the gene annotation database of the species was used with Cufflinks 405 v2.1.1 58 . For differential expressed gene (DEG) analysis, gene level count data were generated by 406 using HTSeq-count v0.6.1p1 59 . The R package called TCC 60 was used to identify DEGs. For 407 comparing tag count data, this package applies robust normalization strategies. Normalization factors 408 were measured by using the iterative DEGES/edgeR method. For correcting errors caused by multiple-409 testing, the DEGs were identified based on the qvalue threshold less than 0.05 61 .   289-300, doi:10.1111/j.2517-6161.1995.tb02031.x (1995). terminated for further analysis. f, Intracellular SCoV2 RNA level in lung tissue isolated from SCoV2-696 infected mice was analysed by qRT-PCR using PCR primers set specific to the SCoV2 N gene. g, 697 Additive anti-SCoV2 effect of vandetanib in combination with remdesivir. HEK293 cells infected with 698 SCoV2 at an MOI of 1 were treated with remdesivir (0.1 nM, about 10,000-fold lower concentration 699 than the IC50 for SCoV2) in the presence or absence of vandetanib (10 μM) for 48 h. Intracellular 700 SCoV2 RNA level was analysed by qRT-PCR using PCR primers set specific to the SCoV2 N gene. P 701 values were calculated by one-way ANOVA (mean ± SD; n =3; *P ≤ 0.0001).    Shin et al.