key: cord-254855-gmy9zyad
authors: He, Sijia; Waheed, Abdul A.; Hetrick, Brian; Dabbagh, Deemah; Akhrymuk, Ivan V.; Kehn-Hall, Kylene; Freed, Eric O.; Wu, Yuntao
title: PSGL-1 inhibits the virion incorporation of SARS-CoV and SARS-CoV-2 spike glycoproteins and impairs virus attachment and infectivity
date: 2020-07-06
journal: bioRxiv
DOI: 10.1101/2020.05.01.073387
sha: 
doc_id: 254855
cord_uid: gmy9zyad

P-selectin glycoprotein ligand-1 (PSGL-1) is a cell surface glycoprotein that binds to P-, E-, and L-selectins to mediate the tethering and rolling of immune cells on the surface of the endothelium for cell migration into inflamed tissues. PSGL-1 has been identified as an interferon-γ (INF-γ)-regulated factor that restricts HIV-1 infectivity, and has recently been found to possess broad-spectrum antiviral activities. Here we report that the expression of PSGL-1 in virus-producing cells impairs the incorporation of SARS-CoV and SARS-CoV-2 spike (S) glycoproteins into pseudovirions and blocks virus attachment and infection of target cells. These findings suggest that PSGL-1 may potentially inhibit coronavirus replication in PSGL-1+ cells.

The ongoing coronavirus disease 2019 (COVID-19) is a global pandemic afflicting more than 10 million people in over 200 countries and territories, resulting in more than 500,000 deaths as of June 30th, 2020. Currently, there are no effective treatments or vaccines. Understanding virushost interactions is critical for developing novel therapeutics and vaccines. P-selectin glycoprotein ligand-1 (PSGL-1, also known as SELPLG or CD162) is a human protein recently identified to possess broad-spectrum antiviral activity (1) . PSGL-1 binds to the selectin family of proteins, P-, E-, and L-selectin (2) , and mediates immune cell tethering and rolling on the surface of endothelium to promote cell migration into inflamed tissues (3) . In the context of viral infection, PSGL-1 has been identified as an IFN-γ-regulated inhibitory factor involved in blocking HIV-1 infectivity (4), and was recently found to possess broad-spectrum antiviral activity (1) , blocking viral infections through steric hindrance of particle attachment to target cells (1, 5) .

The coronavirus spike (S) glycoproteins play an essential role in viral entry by binding the cellsurface receptor on target cells and mediating the fusion between viral and cellular membranes during virus entry (6) . The S protein is also the target of neutralizing antibodies generated by the infected host. Because of its central role in virus infection and adaptive immunity, the S protein is a prime target for the development of antiviral therapeutics and vaccines. In addition to the adaptive arm of the host immune response, viral infections trigger an innate immune response, largely induced by IFN, that sets up an antiviral state. Hundreds of IFN-stimulated genes (ISGs) are induced by viral infection (7) . While the role of some ISGs in blocking the replication of particular viruses has been well established, the vast majority of ISGs have not been characterized. Because of the significance of host innate immunity in viral transmission and 4 replication within and between hosts, there is an unmet need to understand these antiviral inhibitory factors in detail.

Previous studies have demonstrated that PSGL-1 can be incorporated into HIV-1 virions, and its virion incorporation subsequently blocks viral infectivity (1, 5) . To investigate the ability of PSGL-1 to restrict coronavirus infection, we first established a lentiviral vector-based coronavirus pseudovirus infection system (8) , in which the S proteins from either SARS-CoV or SARS-CoV-2 were used to pseudotype lentiviral particles (Fig. 1A ). Using this system, we assembled particles in the presence or absence of PSGL-1 (1), and then used the particles to infect target Vero and Calu-3 cells, which endogenously express the primary SARS-CoV and CoV-2 receptor, angiotensin converting enzyme 2 (ACE2) (9, 10). The expression of PSGL-1 in viral producer cells had a minor (∼ two-fold) effect on the release of SARS-CoV and -CoV-2 pseudovirions ( Fig. 1B and 1C) , consistent with the previous finding that PSGL-1 expression has minimal effects on viral release (1). However, the infectivity of PSGL-1-imprinted SARS-CoV particles was completely abrogated in Vero cells (Fig. 1D) , demonstrating the ability of PSGL-1 to block the infectivity of SARS-CoV S-bearing virions.

We further tested the effect of PSGL-1 on the infectivity of lentiviral particles pseudotyped with the SARS-Cov-2 S protein. We found that particles pseudotyped with SARS-CoV-2 S protein had much lower infectivity than those pseudotyped with SARS-CoV S protein. To resolve this technical issue, we developed a more sensitive reporter system in which a luciferase reporter (Luc) gene was expressed from the HIV-1 LTR in the presence of co-expressed HIV-1 Tat protein (11) (Fig. 1A) . A major advantage of this system is that high-level Luc expression can be achieved upon transactivation by co-expressed Tat protein following viral entry, which minimizes non-specific Luc background from non-productive viral entry (12) . Using this system, 5 we found that the infectivity of the SARS-CoV-2 pseudovirus is also potently inhibited by the expression of PSGL-1 in the virus-producer cells ( Fig. 1E and 1F) . Together, these results demonstrate that PSGL-1 expression in the virus-producer cells severely diminishes the infectivity of virions bearing SARS coronavirus S proteins.

To investigate possible mechanisms, we analyzed the virion incorporation of SARS-CoV S proteins in the presence of PSGL-1. Our previous study showed that PSGL-1 can inhibit the incorporation of the HIV envelope glycoprotein (1). As shown in Fig. 2B , the expression of PSGL-1 in the virus-producer cell also decreased the amount of both SARS-CoV and SARS-CoV-2 S proteins on virions. We and other previously reported that PSGL-1-mediated inhibition of virion infectivity is through steric hindrance of particle attachment to target cells, which does not depend on the presence of viral envelope glycoproteins (1, 5) . We performed a virion attachment assay and observed that the lentiviral particles pseudotyped with SARS-CoV or SARS-CoV-2 S protein produced from PSGL-1-expressing cells were impaired in their ability to attach to target cells (Fig. 2D) . These results demonstrate that the presence of PSGL-1 on virus particles can structurally hinder virion interaction with the target cells even in the presence of remaining S proteins, consistent with previous studies of PSGL-1 and HIV-1 infection (1, 5) .

In this report, we demonstrate that the expression of PSGL-1 in virus-producer cells impairs the infectivity of virions bearing the S protein of either SARS-CoV or SARS-CoV-2, a phenotype shared among several other viruses (e.g., HIV-1, murine leukemia virus, and influenza virus)

found to be sensitive to PSGL-1 restriction (1). PSGL-1 has been suggested to be expressed in certain lung cancer cells (13) , and in lung phagocytes that control the severity of pneumoccal dissemination from the lung to the bloodsstreem (14, 15) . Nevertheless, it remains to be determined whether PSGL-1 is expressed in SARS coronavirus target cells in the lungs, and, if 6 so, whether its expression can impair viral infection. In HIV-1 infection, the viral accessory proteins Vpu and Nef have been shown to antagonize PSGL-1 on CD4 T cells through surface downregulation and intracellular degradation (1, 4) . It also remains unknown whether coronaviruses possess a mechanism for antagonizing PSGL-1.

Virus assembly. The SARS-CoV S and SARS-CoV-2 S protein expression vectors were kindly provided by Gary Whittaker and Nevan Krogan, respectively. A SARS-CoV-2 S protein expression vector was also purchased from Sinobiological. For the production of GFP reporter lentiviral particles pseudotyped with SARS-CoV-S, the SARS-CoV-S expression vector (0.5 µg), pCMVΔR8.2 (7.5 µg), and pLKO.1-puro-TurboGFP (10 µg) were cotransfected with either pCMV3-PSGL-1 (2 µg), or pCMV3-Empty vector (2 µg) as previously described (1) . For the production of luciferase reporter lentiviral particles pseudotyped with SARS-CoV-2-S, the SARS-CoV-2-S expression vector (0.5 µg), pCMVΔR8.2 (7.5 µg), and pLTR-Tat-IRES-Luc (10 µg) were cotransfected with either pCMV3-PSGL-1 (2 µg), or pCMV3-Empty vector (2 µg).

Both of SARS-CoV-S and SARS-CoV-2-S pseudotyped viral particles were produced in HEK293T cells. Virus supernatants were collected at 48 -84 hours post transfection, concentrated by ultracentrifugation, and stored at -80°C.

HIV-1 p24 ELISA. SARS-CoV-S and SARS-CoV-2-S pseudotyped lentiviral particles were quantified using an in-house p24 ELISA kit as previously described (16) .

HIV-1 Env-defective pNL4-3/KFS (1 µg ) and vectors expressing the S protein of either SARS- Data availability All data generated or analyzed during this study are included in this article.

Reagents are available from Y. W. upon request. 

PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells

Expression cloning of a functional glycoprotein ligand for Pselectin

Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P-and E-selectin bound to SLe(X) and PSGL-1

Proteomic profiling of HIV-1 infection of human CD4(+) T cells identifies PSGL-1 as an HIV restriction factor

Virion-incorporated PSGL-1 and CD43 inhibit both cell-free infection and transinfection of HIV-1 by preventing virus-cell binding

Coronavirus membrane fusion mechanism offers a potential target for antiviral development

Interferons: Reprogramming the Metabolic Network against Viral Infection

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites

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

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2

Is Blocked by a Clinically Proven Protease Inhibitor

Tat controls transcriptional persistence of unintegrated HIV genome in primary human macrophages

Development of a nonintegrating Rev-dependent lentiviral vector carrying diphtheria toxin A chain and human TRAF6 to target HIV reservoirs

Activated platelets interact with lung cancer cells through P-selectin glycoprotein ligand-1

Peritoneal macrophages express both Pselectin and PSGL-1

PSGL-1 on Leukocytes is a Critical Component of the Host Immune Response against Invasive Pneumococcal Disease

HIV envelope-CXCR4 signaling activates cofilin to overcome cortical actin restriction in resting CD4 T cells

Left panel; in the absence of PSGL-1 in the virus-producer cell, virions bearing S protein bind the ACE2 receptor and infect the target cell. Right panel; expression of PSGL-1 in the virus-producer cell results in diminished S protein incorporation, and PSGL-1 incorporation into virions sterically blocks virus binding to target cells

Competing interests A provisional patent applications pertaining to the results presented in this paper has been filed by George Mason University.