key: cord-324344-dxuabscn
authors: Zhao, Xuesen; Zheng, Shuangli; Chen, Danying; Zheng, Mei; Li, Xinglin; Li, Guoli; Lin, Hanxin; Chang, Jinhong; Zeng, Hui; Guo, Ju-Tao
title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2
date: 2020-04-05
journal: bioRxiv
DOI: 10.1101/2020.04.02.021469
sha: 
doc_id: 324344
cord_uid: dxuabscn

C3A is a sub-clone of human hepatoblastoma HepG2 cell line with the strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ADAP2, GILT and LY6E, three cellular proteins with known activity of interfering virus entry, expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-OC43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFITM3 restriction of human coronavirus entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a distinct mechanism. Importance Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control by host innate and adaptive immune responses. In the last decade, several interferon inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as host factors to facilitate the entry of several human pathogenic viruses, including human immunodeficiency virus, influenza A virus and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.

OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into 28 C3A cells. In an effort to search for the host cellular protein(s) mediating the differential 29 susceptibility of the two cell lines to HCoV-OC43 infection, we found that ADAP2, GILT and 30 LY6E, three cellular proteins with known activity of interfering virus entry, expressed at 31 significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression 32 of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-OC43. While 33 overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-34 OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to 35

HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry 36 mediated by the envelope spike proteins of other human coronaviruses, including the currently 37 pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or 38 amphotericin treatment significantly neutralized the IFITM3 restriction of human coronavirus 39 entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The 40 work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that 41 controls CoV infection and pathogenesis via a distinct mechanism. 42 LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, 48 LY6E was recently identified as host factors to facilitate the entry of several human pathogenic 49 viruses, including human immunodeficiency virus, influenza A virus and yellow fever virus. (7, 8) , with the mortality rate of 10%, 30% and 1 to 2%, 62 respectively (9, 10). No vaccine or antiviral drug is currently available to prevent CoV infection 63 or treat the infected individuals. The cross-species transmission of zoonotic CoVs presents a 64 continuous threat to global human health (11, 12) . Therefore, understanding the mechanism of 65 CoV infection and pathogenesis is important for the development of vaccines and antiviral 66 agents to control the current COVID-19 pandemics and prevent future zoonotic CoV threats. 67

CoV entry into host cells, a process to deliver viral nucleocapsids cross the plasma 68 membrane barrier into the cytoplasm, is the key determinant of virus host range and plays a 69 critical role in zoonotic CoV cross-species transmission (2, 13). The entry process begins by the 70 binding of viruses to their specific receptor on the plasma membrane, which triggers endocytosis 71 to internalize the viruses into the endocytic vesicles. The cleavage of viral envelope spike 72 proteins by endocytic proteases and/or endosomal acidification triggers the conformation change 73 of spike protein to induce the fusion of viral envelope with endocytic membrane and release 74 nucleocapsids into the cytoplasm to initiate viral protein synthesis and RNA replication. While 75 angiotensin-converting enzyme 2 (ACE2) is the bona fide receptor for SARS-CoV, SARS-CoV-76 and CD13 (also known as aminopeptidase N) as their receptor, respectively (17, 18). However, 78

HCoV-OC43 and HCoV-HKU1 bind to 9-Oacetylated sialic acids via a conserved receptor-79 binding site in spike protein domain A to initiate the infection of target cells (19) . As the key 80 determinant of cell tropism, host range, and pathogenesis, CoV entry is primarily controlled by 81 interactions between the spike envelope glycoprotein and host cell receptor as well as the 82 susceptibility of spike glycoprotein to protease cleavage and/or acid-induced activation of 83 membrane fusion (20, 21) . For instance, SARS-CoV can use ACE2 orthologs of different animal 84 species as receptors (22-26) and the efficiency of these ACE2 orthologs to mediate SARS-CoV 85 cell entry is consistent with the susceptibility of these animals to . 86

In addition, expression of endosomal cathepsins, cell surface transmembrane proteases 87 (TMPRSS), furin, and trypsin differentially modulates the entry of different human CoVs (31-88 35 demonstrated that IFITM1, IFITM1-EX2 and IFITM3-EX2 modulated HCoV-OC43 envelope 146 proteins mediated entry in a similar extent in the two cell lines (Fig. 3E) . Accordingly, we 147 concluded that IFITM proteins were not responsible for the observed differential susceptibility of 148 the two hepatoma cell lines to HCoV-OC43 infection. In this study, we further demonstrated that AmphoB treatment also efficiently attenuated the 215 restriction of IFITM3 on the infection of SARS-CoVpp, MERS-CoVpp, HCoV-NL63pp, HCoV-216 229Epp and IAVpp, but not LASVpp (Fig. 8A) . However, AmphoB treatment altered neither the 217 restriction activity of LY6E on the infection of human CoV spike protein-pseudotyped 218 lentiviruses nor the enhancement of LY6E on IAVpp infection (Fig. 8B) . These results strongly 219 imply that LY6E modulates virus entry via a distinct mechanism. 220 221 DISCUSSION 222 223 LY6E was initially identified as a cell surface marker to discriminate immature from 224 mature thymocytes subsets (53). The primary function of LY6E has been associated with 225 immune regulation, specifically in modulating T cell activation, proliferation, development (54). 226

In addition to lymphocytes, LY6E mRNA can also be detected in liver, spleen, uterus, ovary, 227 lung, and brain and its expression can be induced by type I IFN in a cell-type specific manner 228 Second, in addition to GPI anchor, the evolutionally conserved amino acid residue L36 is 256 also required for both the enhancement and restriction of virus entry into target cells by LY6E 257 (Fig. 6) (48) . It can be speculated that this specific residue may mediate an interaction with other 258 cellular membrane proteins to module viral entry. The fact that LY6E enhances viral infectivity 259 in a cell type-specific manner, with the strongest phenotype in cells of fibroblast and monocytic 260 lineages (48), does indicate the involvement of other host cellular factors. Variations in the 261 abundance of expression, as well as the localization of LY6E and its associated proteins or lipids, 262 may explain the differential effects of LY6E on the infection of different viruses in different cell 263 types ( Fig. 4 and 7) . However, LY6E enhancement of RNA virus infection appears to be 264 independent of type I interferon response and other ISG expression (48) CoV-2pp and HCoV-NL63pp, that share the ACE2 receptor, does not support such a hypothesis 282 (Fig. 4) . 283

Finally, the findings that LY6E inhibits human CoV entry cannot be evaded by ectopic 284 expression of membrane-associated serine protease TMPRSS2 and compromised by AmphoB 285 treatment strongly indicate that LY6E modulates virus entry via a distinct mechanism from that 286 IFITM proteins do (Figs. 7 and 8) . Specifically, inhibition of TMPESS2-enhanced CoV entry 287 implies that LY6E most likely blocks virus entry at plasma membrane or in early endosomes. 288

Moreover, IFITMs impede viral fusion by decreasing membrane fluidity and curvature (37). 289

AmphoB can bind cholesterol in cell membranes to increase membrane fluidity and planarity and 290 consequentially rescue IFITM inhibition of virus entry (52). Interestingly, AmphoB only 291 neutralize the antiviral effects of IFITM2 and IFITM3, but has little effect on IFITM1 restriction 292 of virus entry (52). While IFITM1 is predominantly located in the plasma membrane or early 293 endosomes, IFITM2 and 3 are mainly localized in the later endosomes and lysosomes. Due to 294 their differential subcellular localization, IFITM1 mainly restricts the viruses that enter the cells 295 at cell surface or in the early endosomes, such as parainfluenza viruses and hepatitis C virus (59, 296 60), IFITM2 and 3 primarily restrict the infection of viruses that enter the cells at later 297 endosomes and/or lysosomes (43, 61, 62). Because AmphoB is endocytosed quite rapidly 298 leading to its concentration in the late endosomes and lysosomes, it more efficiently alleviates 299 the effect of IFITM2 and 3, but not IFITM1, on virus entry (52). Similarly, the failure of 300

AmphoB to attenuate the antiviral effects of LY6E against human CoVs is most likely due to its 301 predominant cell surface localization and inhibition of an early step of CoV entry. 302

In summary, while it is very interesting to know that LY6E is capable of modulating the 303 entry of many RNA viruses, we only begin to uncover the mechanism of this fascinating host 304 factor and define its pathobiological role in virus infection (41, 63). Further understanding the 305 role and mechanism of LY6E in viral infections will establish a scientific basis for development 306 of therapeutics to harness its function for the treatment of viral diseases. Real-time RT-PCR. HCoV-OC43 RNA was quantified by a qRT-PCR assay described 390 previously (42). To determine the level of ISG mRNA, total cellular RNA was extracted using 391

TRIzol reagent (Invitrogen) and the same amount of total cellular RNA was reverse-transcribed 392 with SuperScript III kit ((Invitrogen). Quantitative RT-PCR was performed using iTaq universal 393 SYBR Green Supermix (Bio-Rad) with the following primers: LY6E, 5´-394 GTACTGCCTGAAGCCGACCATC-3´ and 5´-AGATTCCCAATGCCGGCACTAG-3´; 395 ADAP2, 5´-AGCTGTCATCAGCATTAAG-3´ and 5´-ACTATCTCCTTCCCACTTTC-3´; 396 GILT, 5´-AATGTGACCCTCTACTATGAAG-3´ and 5´-397 ACGCTGGTGCCCTACGGAAACG-3´; GAPDH, 5´-GAAGGTGAAGGTCGGAGTCAAC-3 398 ´ and 5´-CAGAGTTAAAAGCAGCCCTGGT-3´. Gene expression was calculated using the 2 -△ 399 △ CT method, normalized to GAPDH as described previously (31, 40) . shRNA targeting LY6E mRNA. The level of intracellular LY6E expression was determined by 741

Western blot using a rabbit polyclonal antibody against LY6E. β-actin served as a loading 742

control. (B) HepG2 cells stably expressing the scramble shRNA or LY6E specific shRNA were 743 infected with HCoV-OC43 at an MOI of 1.0. Cells were harvested at 24 hpi and intracellular 744 viral RNA was quantified by qRT-PCR assay and presented as copies per 100 ng total RNA. 745

Error bars indicate standard deviations (n = 4). (C to F) C3A or A549 cells were stably 746 transduced with an empty retroviral vector (pQCXIP) or retroviral vector expressing LY6E and 747

infected with HCoV-OC43 at the indicated MOI. The expression of LY6E in the cell lines was 748 confirmed by a western blot assay. β-actin served as a loading control (C and E). The cells were 749

fixed at 24 hpi. The infected cells were visualized by IF staining of HCoV-OC43 N protein (red). 750

Cell nuclei were visualized by DAPI staining (D and F). 

Coronavirus Host Range Expansion 426 and Middle East Respiratory Syndrome Coronavirus Emergence: Biochemical 427 Mechanisms and Evolutionary Perspectives

A novel coronavirus 432 associated with severe acute respiratory syndrome

Characterization of a novel coronavirus associated with severe acute respiratory 439 syndrome

441 Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia

Genomic characterization of a newly discovered coronavirus associated with acute 446 respiratory distress syndrome in humans

A new coronavirus associated with human respiratory disease in China

A Novel Coronavirus from Patients with Pneumonia 459 in China

Genomic characterisation and epidemiology of 464 2019 novel coronavirus: implications for virus origins and receptor binding

Receptor recognition by novel 471 coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS

474 Mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range 475 expansion in human airway epithelium

Angiotensin-478 converting enzyme 2 is a functional receptor for the SARS coronavirus

481 Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus 482 receptor for cellular entry

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a 486 Clinically Proven Protease Inhibitor

Dipeptidyl peptidase 4 is a functional receptor for the emerging 490 human coronavirus-EMC

Involvement of 492 aminopeptidase N (CD13) in infection of human neural cells by human coronavirus 229E

Human 496 coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved 497 receptor-binding site in spike protein domain A

Host cell proteases: Critical determinants of 502 coronavirus tropism and pathogenesis

Rhesus 504 angiotensin converting enzyme 2 supports entry of severe acute respiratory syndrome 505 coronavirus in Chinese macaques

Expression of feline angiotensin 507 converting enzyme 2 and its interaction with SARS-CoV S1 protein

Mustela vison ACE2 510 functions as a receptor for SARS-coronavirus

GILT restricts the cellular entry mediated by the envelope 559 glycoproteins of SARS-CoV, Ebola virus and Lassa fever virus

Interferon-inducible 563 cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-564 hydroxycholesterol

ADAP2 Is an

Interferon Stimulated Gene That Restricts RNA Virus Entry

Emerging Role of LY6E in Virus-Host Interactions. Viruses 11

Interferon 569 induction of IFITM proteins promotes infection by human coronavirus OC43

Selecting Cells for Bioartificial Liver Devices 579 and the Importance of a 3D Culture Environment: A Functional Comparison between the 580 HepaRG and C3A Cell Lines

CD4-Dependent Modulation of HIV-1 Entry by LY6E. J 582 Virol 93

Interferon-inducible LY6E Protein Promotes HIV-1 584 Infection

Inhibition of human immunodeficiency virus type 1 assembly and release by the 590 cholesterol-binding compound amphotericin B methyl ester: evidence for Vpu 591 dependence

Virus entry, assembly, budding, and membrane rafts

Amphotericin B 598 increases influenza A virus infection by preventing IFITM3-mediated restriction

Thymic shared antigen-1. A novel thymocyte marker discriminating immature from 602 mature thymocyte subsets

Modulation of TCR-mediated signaling pathway by thymic shared 605 antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2)

A Cell Fusion-Based Screening Method Identifies 608 Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse 609

Endogenous Retroviral Envelope Syncytin-A

Deletion 611 of the Syncytin A receptor Ly6e impairs syncytiotrophoblast fusion and placental 612 morphogenesis causing embryonic lethality in mice

IFITM 616 proteins inhibit placental syncytiotrophoblast formation and promote fetal demise

Interferon-induced transmembrane proteins inhibit cell fusion mediated 620 by trophoblast syncytins

Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3

IFITM1 is a tight junction protein that inhibits hepatitis C 626 virus entry

Distinct patterns of IFITM-mediated 630 restriction of filoviruses, SARS coronavirus, and influenza A virus

IFITM3 inhibits influenza A virus infection by 634 preventing cytosolic entry

Relating GPI-Anchored Ly6 Proteins uPAR and CD59 636 to Viral Infection. Viruses 11

Interferon-641 induced cell membrane proteins, IFITM3 and tetherin, inhibit vesicular stomatitis virus 642 infection via distinct mechanisms

Identification of five interferon-induced cellular 645 proteins that inhibit west nile virus and dengue virus infections

Small molecule inhibitors of ER alpha-glucosidases are active against 649 multiple hemorrhagic fever viruses

651 Characterization of the spike protein of human coronavirus NL63 in receptor binding and 652 pseudotype virus entry

Inhibition of endoplasmic reticulum-resident 655 glucosidases impairs severe acute respiratory syndrome coronavirus and human 656 coronavirus NL63 spike protein-mediated entry by altering the glycan processing of 657 angiotensin I-converting enzyme 2

Vpr is required for efficient 662 replication of human immunodeficiency virus type-1 in mononuclear phagocytes

An 666 interferon-beta promoter reporter assay for high throughput identification of compounds 667 against multiple RNA viruses

C3A cells support more efficient entry of lentiviral particles pseudotyped with 690

HCoV-OC43 envelope proteins than HepG2 cells. HepG2 and C3A cells were infected with 691

Luciferase activities were determined at 692 72 hpi. Relative infection represents the luciferase activity from C3A normalized to that of 693 HepG2 cells. Error bars indicate standard deviations (n = 6). ** indicates p <0

Cell nuclei were visualized by DAPI staining. (B) HCoV-OC43 NP and 705 IFITM were determined by Western blot assays. β-actin served as a loading control

Error bars indicate standard deviations (n = 4). (E) HepG2 and C3A stably 709 transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing IFITM1, 710 IFITM1-EX2 or IFITM3-EX2 were infected with HCoV-OC43pp

HepG2 cells transduced with empty vector (pQCXIP)

The amino acid sequence alignment of LY6E from multiple vertebrate 755 species is conducted and "three finger-fold" structure is highlighted with black box. The 756 conserved L36 as well as GPI anchor and N99 glycosylation sites are indicated (B) Flp-In T-Rex 757 293-derived cell lines expressing a control protein CAT, wild-type or mutant LY6E were 758 cultured in the absence or presence of tet for 24 h. Intracellular LY6E expression were detected 759 by a Western blot assay. β-actin served as a loading control. (C) Flp-In T-Rex 293-derived cell 760 lines expressing the wild-type or mutant LY6E were cultured in the absence or presence of tet for 761 24 h. The cells were then infected with the indicated pseudotyped lentivirus

Flp-In T-Rex 788 293-derived cell line expressing IFITM3 (A) or LY6E (B) were cultured in the absence or 789 presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus in 790 the presence or absence of 1μM AmphoB. Luciferase activity was measured at 48 hr post-791 infection

001, compared to mock treatment