key: cord-0740116-pul32uuo authors: Zhang, Fei; Li, Wan; Feng, Jian; Ramos da Silva, Suzane; Ju, Enguo; Zhang, Hu; Chang, Yuan; Moore, Patrick S.; Guo, Haitao; Gao, Shou‐Jiang title: SARS‐CoV‐2 pseudovirus infectivity and expression of viral entry‐related factors ACE2, TMPRSS2, Kim‐1, and NRP‐1 in human cells from the respiratory, urinary, digestive, reproductive, and immune systems date: 2021-08-04 journal: J Med Virol DOI: 10.1002/jmv.27244 sha: 08ec38e7bf03bae6880976afa71eb91644448d0e doc_id: 740116 cord_uid: pul32uuo Infection by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes a wide spectrum of syndromes involving multiple organ systems and is primarily mediated by viral spike (S) glycoprotein through the receptor‐binding domain (RBD) and numerous cellular proteins including ACE2, transmembrane serine protease 2 (TMPRSS2), kidney injury molecule‐1 (Kim‐1), and neuropilin‐1 (NRP‐1). In this study, we examined the entry tropism of SARS‐CoV‐2 and SARS‐CoV using S protein‐based pseudoviruses to infect 22 cell lines and 3 types of primary cells isolated from respiratory, urinary, digestive, reproductive, and immune systems. At least one cell line or type of primary cell from each organ system was infected by both pseudoviruses. Infection by pseudoviruses is effectively blocked by S1, RBD, and ACE2 recombinant proteins, and more weakly by Kim‐1 and NRP‐1 recombinant proteins. Furthermore, cells with robust SARS‐CoV‐2 pseudovirus infection had strong expression of either ACE2 or Kim‐1 and NRP‐1 proteins. ACE2 glycosylation appeared to be critical for the infections of both viruses as there was a positive correlation between infectivity of either SARS‐CoV‐2 or SARS‐CoV pseudovirus with the level of glycosylated ACE2 (gly‐ACE2). These results reveal that SARS‐CoV‐2 cell entry could be mediated by either an ACE2‐dependent or ‐independent mechanism, thus providing a likely molecular basis for its broad tropism for a wide variety of cell types. supporting a direct role of SARS-CoV-2 infection in causing the complex pathologies of COVID-19. 10 The tropism of SARS-CoV-2 infection for various cell types is key to understanding these pathologic and clinical COVID-19 features. Numerous cell lines are permissive to SARS-CoV-2 infection in cell culture. Vero E6, an African green monkey kidney cell line, has been widely used in SARS-CoV-2 culture. 11, 12 Several cell lines from the digestive system (Caco-2) and liver system (Huh-7) are permissive to SARS-CoV-2 infection, 13 and Calu-3 cells isolated from the human respiratory system have been used in SARS-CoV-2 infection studies. [14] [15] [16] Interestingly, primary human alveolar epithelial cells (AECs) and the lung A549 cell line are refractory to infection by both SARS-CoV-2 and SARS-CoV. 13, 17, 18 Numerous cellular proteins have been identified to mediate SARS-CoV-2 entry including angiotensin-converting enzyme 2 (ACE2), 19, 20 transmembrane serine protease 2 (TMPRSS2), 21 kidney injury molecule-1 (Kim-1), a biomarker for human renal proximal tubule injury, 22,23 and neuropilin-1 (NRP-1), which binds to furin-cleaved substrates. 24 posttransfection, centrifuged at 3,000 rpm for 10 min, aliquoted, and stored at −80°C for later use. To investigate the susceptibility of various cells to pseudoviruses, human Fc protein (hFc, AG100, Sigma). The supernatant containing the pseudovirus was mixed with an equal volume of a specified recombinant protein diluted in medium to achieve the specified concentration at 37°C for 1 h, and used for the infection assay. Protein lysates of Huh7 were prepared in NP40 Cell Lysis Buffer In the infection assay, luciferase activity higher than the mean of mock-infected cells plus 5 times of SEM was considered as Entry of SARS-CoV-2 into cells is mediated by the S protein through RBD interaction with cellular receptor(s). 13, 14 To demonstrate the specificity of the pseudovirus infections, we performed blocking assays using S1 and RBD fusion recombinant proteins in Huh-7 cells, which had the highest pseudovirus infection levels ( Figure 1A ). Both S1 and RBD recombinant proteins effectively blocked the infection of SARS-CoV-2 pseudovirus in a dose-dependent manner, reducing luciferase signals by >60 to >95% at the highest dose of 25 µg/ml, respectively (Figure 2A,B) . In contrast, a control hFc recombinant protein did not block any We then examined expression levels of ACE2, TMPRSS2, Kim-1, and NRP-1 proteins by Western blotting ( Figure 4A ). Despite some discrepancies, the protein levels were in general in agreement with the mRNA levels of genes encoding these proteins. For ACE2 protein, we detected two bands representing the glycosylated and unglycosylated forms (gly-ACE2 and ungly-ACE2) with the expected molecular mass sizes of~85 kD and 120 kD, respectively ( Figure 4A ). 31 Treatment of cell lysate with We examined the correlation between SARS-CoV-2 pseudovirus infectivity and expression levels of ACE2, TMPRSS2, Kim-1, and NRP-1 proteins ( Figure 1A ). SARS-CoV-2 pseudovirus infectivity had a positive correlation with the gly-ACE2 protein level, approaching statistical significance (r = 0.3840, p = 0.0581), but not with unglycosylated ACE2 (r = 0.1651, p = 0.4303) ( Figure 6A,B) . proteins in a wide range of parenchymal and immune cells. 10 Results of these studies suggest that SARS-CoV-2 could infect a wide range of cell types. In the current study, we investigated the cell tropism of SARS-CoV-2 using a pseudovirus to infect 22 cell lines and 3 types of primary cells isolated from respiratory, urinary, immune, digestive, and reproductive systems ( Figure 1A and Table 3 Entry of SARS-CoV-2 and SARS-CoV is primarily mediated by viral S1 protein through its interaction with cellular receptors. 39 Our 10 Indeed, among the cell lines and primary cell types that were positively infected by SARS-CoV-2 pseudovirus, we also found that A549 and HRC45 cells had extremely weak levels of ACE2 protein expression ( Figure 4A ). Nevertheless, we found a trend of positive correlation of SARS-CoV-2 pseudovirus infectivity with the level of gly-ACE2 protein but not ungly-ACE2 protein ( Figure 6A,B) . It has been reported that in engineered human tissue, SARS-CoV-2 infection was inhibited by soluble human ACE2. 48 Our results showed that recombinant ACE2 protein effectively blocked the infectivity of SARS-CoV-2 pseudovirus in Huh-7 cells and that the ACE2 inhibitory effect occurred in a lower concentration than that reported in a previous study 48 ( Figure 2C ). These results support an important role of ACE2 protein, particularly gly-ACE2 protein, in SARS-CoV-2 infection of ACE2dependent cells. On the other hand, we found a statistically significant positive correlation of SARS-CoV pseudovirus infectivity with the levels of gly-ACE2 protein but not ungly-ACE2 protein. Hence, compared with SARS-CoV, SARS-CoV-2 infection is less dependent on gly-ACE2, which might explain its promiscuous broad tropism and more infectious nature. 10 In fact, the dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike has been reported. 49 Specifically, the glycans at the N90 and N322 glycosylation sites had opposite effects on S protein binding. 49 Interestingly, A549 and HRC45 cells had a robust SARS-CoV- The "red" dots represent cell lines/types that were infected by the pseudoviruses. The "green" dots represent cell lines/types that were not infected by the pseudoviruses. 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