key: cord-0991813-nrhg5krp
authors: Yang, Hang; Yuan, Huijun; Zhao, Xiaohui; Xun, Meng; Guo, Shangrui; Wang, Nan; Liu, Bing; Wang, Hongliang
title: Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells
date: 2022-03-07
journal: Virol Sin
DOI: 10.1016/j.virs.2022.03.003
sha: 29404a9fbe530e58ed38de06ceffee8f6acd9758
doc_id: 991813
cord_uid: nrhg5krp

The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor-human angiotensin converting enzyme 2 (ACE2). Here, by using lentivirus based pseudotypes bearing spike protein, we demonstrated that entry of SARS-CoV-2 into host cells was dependent on clathrin-mediated endocytosis, and phosphoinositides played essential roles during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 were not required for efficient virus entry. Finally, we showed that the current predominant Delta variant, although with high infectivity and high syncytium formation, also entered cells through clathrin-mediated endocytosis. These results provide new insights into SARS-CoV-2 cellular entry and present proof of principle that targeting viral entry could be an effective way to treat different variant infections.

mutant assay, 293T cells were transfected with ACE2 or the indicated ACE2 mutant and 30 h 172 later, cells were split for immunoblotting and pseudovirus transduction. For cells transduced 173 with GFP pseudovirus, photos were taken at 56 h post-inoculation. For virus entry inhibition 174 studies, cells were pretreated with indicated concentrations of inhibitors for 1 h at 37 °C 175 before pseudovirus inoculation. NanoLuc luciferase activity was measured as described 176 above. 177

Immunoblotting and qPCR were carried out as previously reported (Yang et al., 2021) . Briefly, 179 for immunoblotting, cells were lysed with LDS sample buffer (Thermo Fisher Scientific) and 180 subjected to SDS-PAGE followed by transferring to a PVDF membrane. The blots were then 181 probed with indicated antibodies and bands were visualized with SuperSignal TM West Femto 182

Maximum Sensitivity Substrate (Thermo Fisher Scientific). For S protein incorporation test, 183 pseudoviruses were first ultracentrifuged as described above and then subjected to 184 SDS-PAGE. To quantify phosphatidylinositol 4-kinase β (PI4KB) mRNA, total RNAs from 185 control or shRNA transduced cells were extracted with GeneJET RNA Purification Kit and 186 then reversed transcribed with random primer and RevertAid Reverse Transcription kit 187 (Thermo Fisher Scientific). Quantification was performed by real-time quantitative PCR with 188 SYBR Mix (Genstar qPCR Master Mix) using specific primers listed in Supplementary Table  189 S2. 190

Immunofluorescence staining (except for ACE2 staining) was performed as described in 192 (Yang et al., 2021) . Images were taken with a Nikon C2 laser scanning confocal microscope 193 in sequential scanning mode to limit crosstalk between fluorochromes. Quantitative 194 colocalization was analyzed by NIH Image J with JACoP plugin (Bolte and Cordelieres, 195 2006). To stain ACE2 and mutants expression on cell surface, cells were fixed without any 196 detergent treatment to keep cell membrane intact. Cells were then stained with anti-ACE2 197 antibody followed by Alexa-488 goat anti-rabbit secondary antibody.

pLKO.1-Puro based shRNA or pLentiCRISPR V2 based sgRNA lentiviral vectors and 202 supernatants were harvested and used for transduction. sgRNA sequences and shRNA 203 sequences used were listed in Supplementary Table S3 . Cells transduced with lentiviral 204 particles were then selected with puromycin for stable cell lines. 205

All values represent means ± standard deviations and represent the results of a minimum of 207 three independent experiments. Where applicable, the two-tailed Student's t-test was used to 208 compare the means of control and experimental groups. 209

Pseudovirus has been widely used to mimic the entry of real virus and is a powerful tool for 213 studying early events in the life cycle of a virus. We here employed a lentivirus-based 214 pseudovirus to study the entry of SARS-CoV-2. A codon-optimized cDNA encoding the S 215 protein was employed, and the C-terminal last 19 amino acids, which contain an endoplasmic 216 reticulum retention signal were replaced with FLAG tag (Supplementary Fig. S1A ). We first 217 showed that codon-optimization enhanced S protein expression in 293T cells (Supplementary 218 Fig. S1B ). In addition, consistent with previous reports (Hoffmann et al., 2020; Ou et al., 219 2020; Walls et al., 2020), the S protein was processed and two major bands were observed, 220 reflecting the full-length and cleaved S proteins respectively ( Supplementary Fig. S1B ). To 221 confirm S protein was efficiently incorporated into the pseudoviral particles, S 222 protein-bearing pseudoviruses (S pseudoviruses) were pelleted by ultracentrifugation and 223 tested against anti-spike antibody (Fig. 1A) . The results showed that S protein was 224 incorporated into viral particles and the majority of S proteins on pseudovirus were also 225 cleaved, further suggesting the S protein was efficiently processed in host cells. 226

Next, we tested whether these S pseudoviruses were able to transduce host cells. For this 227 purpose, we infected 293T, Vero E6, Huh 7 and 293T cells overexpressing ACE2 228 by S pseudoviruses, although at low level compared to other cells. On the other hand, 232 overexpression of ACE2 in 293T cells significantly enhanced S pseudovirus infection. 233

Immunoblotting showed that there was low level of ACE2 expression in 293T cells, while 234 293T-ACE2 had significantly enhanced ACE2 expression (Fig. 1C) . These results revealed a 235 correlation between ACE2 expression and S pseudovirus transduction, suggesting 236 SARS-CoV-2 utilizes ACE2 as a receptor. Although both Vero E6 and Huh 7 cells had 237 moderate ACE2 expression and could be transduced by S pseudovirus efficiently, Huh 7 gave 238 more than two-fold luciferase activity than Vero E6 cells ( Fig. 1B and C) . Similarly, 239 GFP-expressing pseudoviruses also transduced the above cells lines successfully and Huh 7 240 cells showed more positive cells than Vero E6 cells (Fig. 1D ). Therefore, Huh 7 was chosen 241 for the following viral entry studies. 242

The entry of SARS-CoV-2 into host cells requires the activation of its surface spike protein 244 by cell surface serine protease TMPRSS2 or endosomal cysteine proteases cathepsin B/L 245 (Hoffmann et al., 2020). We next tested whether SARS-CoV-2 entry into Huh 7 cells could be 246 blocked by TMPRSS2 inhibitor, camostat mesylate or cathepsin inhibitor, E-64d. Fig. 2A  247 showed that camostat mesylate did not interfere with SARS-CoV-2 entry into Huh 7 cells, 248 which was blocked by E-64d in a dose-dependent manner. Cellular ATP content measurement 249 showed that neither drug caused cytotoxicity ( Supplementary Fig. S2A ). Consistent with this, 250 we found that Huh7 cells had relatively low TMPRSS2 expression compared to Caco2 cells 251 ( Supplementary Fig. S2B ), which has been shown to be sensitive to camostat mesylate 252 (Hoffmann et al., 2020). These results suggested that SARS-CoV-2 could use endosomal 253 proteases for spike priming during Huh 7 cell infection. We then tested whether SARS-CoV-2 254 could enter cells via endocytosis. In contrast to direct membrane fusion, endocytosis is 255 thought to be pH-dependent (White and Whittaker, 2016) . When Huh 7 cells were pretreated 256 with lysosomotropic agents, like chloroquine, ammonium chloride or bafilomycin A1, 257 significant decrease of transduction was observed for both VSV-G and SARS-CoV-2 S 258 pseudoviruses without causing any cytotoxicity (Fig. 2B, Supplementary Fig. S2C However, neither drug inhibited the S pseudovirus or VSV-G pseudovirus transduction (Fig.  287 3A, 3B), indicating that SARS-CoV-2 entry of Huh 7 cells was independent of pseudovirus could infect Huh 7 cells in the absence of CAV1, suggesting the viral entry was 292 independent of caveolae. This finding was further corroborated by the immunostaining 293 experiment, which showed little colocalization between SARS-CoV-2 spike protein and 294 CAV1 (Fig. 3E , Pr = 0.064). Taken together, these results indicate that SARS-CoV-2 entry is 295 independent of caveolae-mediated endocytosis. 296

PI is known to be involved throughout the process of endocytosis. Of note, PI(4,5)P2, PI4P 298 and PI(3,4)P2 are essential molecules in endocytosis. We found that overexpression of either 299 INPP5E, which converted PI(4,5)P2 to PI4P, or the sac1 phosphatase, which dephosphorylated 300 PI4P (Fig. 4A, Supplementary Fig. S4A ), inhibited S pseudovirus transduction without 301 cytotoxicity (Fig. 4B, Supplementary Fig. S4B ), suggesting that PI4P and PI(4,5)P2 were 302 involved in SARS-CoV-2 entry. Importantly, pretreatment of Huh 7 cells with 303 phosphatidylinositol kinase inhibitors, PIK93 or wortmannin, inhibited S pseudovirus 304 transduction at concentrations that effectively inhibited PI4-kinase activity (Fig. 4C , 305

Supplementary Fig. S4D and E), suggesting that PI4 kinase was involved in SARS-CoV-2 306 infection. As PI4KB has been shown to be involved in SARS-CoV infection (Yang et al., 307 2012), we next tested whether it is also required for SARS-CoV-2 infection. For this purpose, 308 we first knocked down PI4KB expression with shRNAs, and qPCR results showed that all 309 three shRNAs could efficient target PI4KB expression ( Fig. 4D open bars, Supplementary Fig.  310 S4C). When these cells were infected with S pseudovirus, we found that PI4KB knockdown 311 led to significant decrease in SARS-CoV-2 transduction (Fig. 4D , black bars), suggesting 312 PI4KB was required for SARS-CoV-2 infection. Fig. S6A ). We found this Spikedelta was present predominantly in the cleaved 352 form compared to wild-type spike, while spike that has mutation in the furin cleavage site 353 (Spikefur/mut) mainly presented as uncleavaged form (Fig. 6A) , suggesting the introduction of 354 P681R mutation enhanced spike cleavage. Consistent with the fact that Delta variant has 355 higher transmissibility, increased infectivity was also observed for Spikedelta mediated cell 356 entry (Fig. 6B ). In addition, Spikedelta also mediated higher cell fusion and syncytium 357 formation than WT Spike, while the Spikefur/mut barely induced any syncytium formation ( Spike with Delta unique mutations, we showed that although this variant has high infectivity 407 and causes increased syncytia formation, it still enters cells through CME. These results 408 provided proof of principle that targeting viral entry could be an effective way to block PI4P and PI4KB have been reported to be essential for SARS-CoV infection (Yang et al., 411 2012) and a recent report showed that PIKfyve inhibitors could block S pseudovirus infection 412 (Ou et al., 2020) , which all suggested that phosphoinositide pathways were important for 413 coronavirus infection. Here we further demonstrated that PI4P and PI(4,5)P2 were essential 414 molecules for SARS-CoV-2 infection and PI4KB was also required for SARS-CoV-2 entry. 

In summary, here we found that the entry of SARS-CoV-2 into host cells was dependent on 425 CME by using lentivirus based pseudotypes bearing spike proteins, and PI played essential 426 roles during this process. Meanwhile, the intracellular domain and the catalytic activity of 427 ACE2 are not required for efficient virus entry. In addition, Delta variant, which has high 428 infectivity and high syncytium formation rate, also enters cells through CME. These results 429 provide new insights into SARS-CoV-2 cellular entry and present proof of principle that 430 targeting viral entry could be an effective way to treat different variant infections. Cleaved and uncleaved spike were indicated. Band densities were quantified with Image J and the ratios of cleaved band density to that of uncleaved band were shown below. B Huh 7 cells transduced with 3 × 10 7 GEq pseudovirus bearing wild Spike, Spikefur/mut, or Spikedelta and luciferase was measured. Values from wild type spike were set to 1. C 293T-ACE2 cells were transfected with construct encoding Spike, Spikefur/mut, or 

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