key: cord-0698352-wa9ep45o
authors: Lin, Zebin; Wu, Zhiming; Mai, Jinlian; Zhou, Lishi; Qian, Yu; Cai, Tian; Chen, Zhenhua; Wang, Ping; Lin, Bin
title: The Nucleocapsid Protein of SARS-CoV-2 Abolished Pluripotency in Human Induced Pluripotent Stem Cells
date: 2020-05-01
journal: bioRxiv
DOI: 10.1101/2020.03.26.010694
sha: 630b77ead07fab4a1b7f4e142ae1677b6ffb4e5b
doc_id: 698352
cord_uid: wa9ep45o

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging across the world, leading to a global mortality rate of 3.4% (estimated by World Health Organization in March 2020). As a potential vaccine and therapeutic target, the nucleocapsid protein of SARS-CoV-2 (nCoVN) functions in packaging the viral genome and viral self-assembly. To investigate the biological effects of nCoVN to human stem cells, genetically engineered human induced pluripotent stem cells (iPSC) expressing nCoVN (iPSC-nCoVN) were generated by lentiviral expression systems, in which the expression of nCoVN could be induced by the doxycycline. The proliferation rate of iPSC-nCoVN was decreased. Unexpectedly, the morphology of iPSC started to change after nCoVN expression for 7 days. The pluripotency marker TRA-1-81 were not detectable in iPSC-nCoVN after a four-day induction. Meanwhile, iPSC-nCoVN lost the ability for differentiation into cardiomyocytes with a routine differentiation protocol. The RNA-seq data of iPSC-nCoVN (induction for 30 days) and immunofluorescence assays illustrated that iPSC-nCoVN were turning to fibroblast-like cells. Our data suggested that nCoVN disrupted the pluripotent properties of iPSC and turned them into other types of cells, which provided a new insight to the pathogenic mechanism of SARS-CoV-2.

Introduction 44

Right now, the COVID-19 pandemic is sweeping the world, causing a huge crisis in public health 45 and economics globally. According to the continuously updated data from World Health 46

Organization, to date, nearly three million infected cases were confirmed, while more than 200,000 47 individuals died because of COVID-19 (https://www.who.int/emergencies/diseases/novel-48 coronavirus-2019). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was 49 proved to be the pathogen of COVID-19, has 79% identity in genomes with severe acute respiratory 50 syndrome coronavirus (SARS-CoV) (Lu et al., 2020) . Twelve coding regions were predicted in 51 SARS-CoV-2, including spike protein, nucleocapsid protein, envelope protein, and membrane 52 protein (Lu et al., 2020; Wu et al., 2020; Xu et al., 2020) . The Cryo-EM structure of spike protein 53 had been determined (Wrapp et al., 2020) , and more and more evidences showed that the spike 54 protein binds human ACE2 to entry into host cells (Hoffmann et al., 2020; Wrapp et al., 2020) , which 55

indicated that SARS-CoV-2 might share similar pathogenic mechanisms with SARS-CoV. Because 56 of the very limited knowledge of SARS-CoV-2, we sought to understand the biology of SARS-CoV-57 2 based on the previous studies about SARS-CoV. 58

As one of the most studied proteins in SARS-CoV, the nucleocapsid protein binds to viral RNA to 59 package the genome in a ribonucleoprotein particle (Chang et al., 2014) . Unlike the spike protein 60 with a certain mutation frequency, the sequence of nucleocapsid protein was more stable (Chinese, 61 2004), which meant it was an ideal target for diagnostic tools ( pathogenic effects in host cells caused by the nucleocapsid protein were also studied. It was reported 64 that the nucleocapsid protein inhibited type I interferon production after virion infected the host cells 65 (Hu et al., 2017) , which was considered as a possible mechanism of immune escape. The 66 nucleocapsid protein inhibited cell cytokinesis and proliferation (Zhou et al., 2008) , and regulated 67 several pathways, such as transforming growth factor-beta signaling (Zhao et al., 2008) , AP-1 signal 68 transduction pathway (He et al., 2003) , and NF-KappaB pathway (Zhang et al., 2007b) . Besides, the 69 nucleocapsid protein was reported as an apoptosis inducer in COS-1 cells ( sorts of stem cells. Figure 1A showed showed that ACE2 protein was located on the cell membrane of iPSC ( Figure 1C ), suggesting that 195 the pluripotent stem cells were the potential targets of SARS-CoV-2. 196

To study whether physiological activities in iPSC were disturbed by nCoVN, a human induced 198 pluripotent stem cell line (iPSC-nCoVN) in which the expression of nCoVN could be modulated by a 199 Tet-On system was generated by a lentiviral expression system. In this system, nCoVN cDNA 200 sequence (with a 6× His Tag coding sequence) was conjugated to puromycin resistance gene through 201 a T2A peptide encoding sequence, and the transcription was relied on the induction of tetracycline or 202 doxycycline (Dox). After puromycin selection, two single cell clones were seeded in separated wells 203 by manual colony-picking. Sequentially, iPSC-nCoVN were divided into two groups: one was 204 induced by Dox for nCoVN expression (Dox), the other was added with DMSO as a control set 205 (DMSO), meanwhile, a GFP-expressed iPS cell line (iPSC-GFP), in which the expression of GFP 206 was modulated by the same Tet-On system, was used as another control set in the following assays 207 (Figure 2A) . 208

The expression of nCoVN was confirmed at the mRNA and protein levels. The transcriptional level 209 of nCoVN was measured by Real-time PCR in iPSC, Dox, and DMSO groups, and nCoVN 210 expression increased about 267-fold in the Dox group compared with the DMSO group ( Figure 2B ). 211

The nCoVN protein was detected by using an anti-6× His Tag antibody in cells from the Dox group 212

(Supplementary Figure 1) . The proliferation rate was compared among iPSC, iPSC-GFP and Dox 213 groups by using a cell counting kit. The absorbance at 450 nm (A450) was measured at 24 hours, 42 214 hours, 48 hours, 60 hours and 72 hours after cell seeding. After three days of cell seeding, Dox group 215 showed a decreased proliferation rate than both of iPSC and iPSC-GFP groups, indicating that 216 nCoVN might hamper the growth and division of iPSC ( Figure 2C ). This observation was consistent 217

with the previous finding about the nucleocapsid protein of SARS-CoV (Zhou et al., 2008) . 218

We continued to induce nCoVN expression in iPSC. The phase-contrast images of iPSC-nCoVN 219 with a 7-day, a 9-day, and an 11-day inductions (Dox) and counterpart controls (DMSO) were shown 220

in Figure 2D . In the DMSO group, a typical morphology of stem cells with high nucleus/cytoplasm 221 ratio and close cell membrane contacts was observed, while iPSC-nCoVN after a 7-day induction 222 started to exhibit endothelial cell morphological features and lower nucleus/cytoplasm ratio. After a 223 14-day induction, most of the cells exhibited distinct shapes from wild-type iPSC, such as neuron-224 like cells, endothelial-like cells and fibroblast-like cells ( Figure 2E ). These data showed that 225 continuous expression of nCoVN caused obvious morphological changes in iPSC. Figure 2A, B) . We traced the expression of TRA-1-81 in iPSC-nCoVN with a 2-day, 233 a 4-day, a 6-day, and an 8-day inductions ( Figure 3A ). On Day 2, TRA-1-81 was still expressed in 234 iPSC-nCoVN; however, from Day 4, TRA-1-81 was not detectable in most of the cells, suggesting 235 that the pluripotent fate of iPSC-nCoVN was determined in the first 4 days. To further test the 236 pluripotency in iPSC and iPSC-nCoVN, we directly differentiated these cells to cardiomyocytes by 237 using a routine protocol, and the differentiation assays were performed under the same conditions. As 238 expected, the differentiation efficiency could reach 60% in iPSC; however, on differentiation day 12, 239

only a very small portion of cells from iPSC-nCoVN were expressed cardiac Troponin T, 240 accompanied by many cell deaths ( Figure 3B, C) . This differentiation assay provided solid evidence 241 that the pluripotency maintenance of iPSC-nCoVN was disrupted by nCoVN. 242

Since the pluripotency lost due to short-term expression of nCoVN, we are extremely interested in 244 the cell fate of iPSC-nCoVN under long-term expression of nCoVN. After a 28-day induction in the 245 stem cell culture medium, some spindle-shaped iPSC-nCoVN, which exhibited a typical fibroblast 246 morphological feature, were observed (Supplementary Figure 2C) . The antibodies against fibroblast 247 markers vimentin, alpha-smooth muscle actin (α-SMA) and S100A4 were used to verify the cell type 248 of these fibroblast-like cells. The results from immunofluorescence assays confirmed that these 249 markers were expressed in nCoVN-expressing cells ( Figure 4A , B, C; Supplementary Figure 2D ). To 250 further investigate the transcriptomic profiles of iPSC-nCoVN under the long-term nCoVN 251 expression, doxycycline-induced iPSC-nCoVN and iPSC-GFP for 30 days were applied to RNA-seq. 252

Through differentially express analysis, iPSC-nCoVN showed a dramatic gene expression change 253

comparing with iPSC-GFP (Supplementary Table) . Totally, 3,080 genes were significantly 254 differentially expressed (FDR<0.05, |log2FoldChange|>3). Among them, the down-regulated genes 255

in iPSC-nCoVN were most significantly enriched with proliferation and stem cell related pathways, 256

including the Yamanaka factors-associated genes, such as POU5F1, LIN28A, NANOG, and SOX2 257

(with a 790-fold, a 2306-fold, a 253-fold, and an 18-fold decrease, respectively) ( Figure 4E ); while 258 the extracellular matrix and extracellular matrix-associated pathway was the most significantly 259 enriched pathway in the up-regulated genes ( Figure 4F ). Next, we used RNA-seq data from the iPSC-GFP samples were clustered with H7 and GM23338, which were the embryonic stem cells 263

(ESC) and iPSC, respectively; while iPSC-nCoVN samples were clustered with multiple kinds of 264 fibroblast ( Figure 4G ). Furthermore, iPSC-nCoVN with a 40-day induction, which were kept 265 culturing in the stem cell medium, were totally differentiated to fibroblast ( Figure 4D ). which was also the possible pathway that nCoVN turned iPSC to fibroblast. 282

The time-course assays showed that the pluripotency marker disappeared in four days after nCoVN 283

expression. This finding might be applied to a cell-based chemical screening model, in which the 284 candidate chemicals with potential ability to halt the iPSC differentiation caused by nCoVN are 285 easily identified. More importantly, SARS-CoV-2 is not necessary in this model, which means it 286 could be used in the routine laboratories and applied to high-throughput equipment with less risk. 287

In addition, how nCoVN breaks the pluripotency maintenance of iPSC is still a riddle. This work is dedicated to all the medical staff who are still fighting against COVID-19 in China. 310

Your efforts make us safer. 

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