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Role of host factors in SARS-CoV-2 entry

  • John P. Evans
    Affiliations
    Center for Retrovirus Research, The Ohio State University, Columbus, Ohio, USA

    Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA

    Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA
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  • Shan-Lu Liu
    Correspondence
    For correspondence: Shan-Lu Liu
    Affiliations
    Center for Retrovirus Research, The Ohio State University, Columbus, Ohio, USA

    Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA

    Department of Microbial Infection and Immunity, The Ohio State University, Columbus, Ohio, USA

    Viruses and Emerging Pathogens Program, Infectious Diseases Institute, The Ohio State University, Columbus, Ohio, USA
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Open AccessPublished:May 27, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100847
      The zoonotic transmission of highly pathogenic coronaviruses into the human population is a pressing concern highlighted by the ongoing SARS-CoV-2 pandemic. Recent work has helped to illuminate much about the mechanisms of SARS-CoV-2 entry into the cell, which determines host- and tissue-specific tropism, pathogenicity, and zoonotic transmission. Here we discuss current findings on the factors governing SARS-CoV-2 entry. We first reviewed key features of the viral spike protein (S) mediating fusion of the viral envelope and host cell membrane through binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2. We then examined the roles of host proteases including transmembrane protease serine 2 and cathepsins in processing S for virus entry and the impact of this processing on endosomal and plasma membrane virus entry routes. We further discussed recent work on several host cofactors that enhance SARS-CoV-2 entry including Neuropilin-1, CD147, phosphatidylserine receptors, heparan sulfate proteoglycans, sialic acids, and C-type lectins. Finally, we discussed two key host restriction factors, i.e., interferon-induced transmembrane proteins and lymphocyte antigen 6 complex locus E, which can disrupt SARS-CoV-2 entry. The features of SARS-CoV-2 are presented in the context of other human coronaviruses, highlighting unique aspects. In addition, we identify the gaps in understanding of SARS-CoV-2 entry that will need to be addressed by future studies.

      Keywords

      Abbreviations:

      ACE2 (angiotensin converting enzyme 2), Cat B/L (cathepsin B/L), CD147 (cluster of differentiation 147), Cyp A/B (cyclophilin A/B), HCoV (human coronavirus), HSPG (heparin sulfate proteoglycan), IFITM (interferon-induced transmembrane proteins), ISG (interferon-stimulated gene), Ly6E (lymphocyte antigen 6E), MERS-CoV (Middle East respiratory syndrome coronavirus), MHV (mouse hepatitis virus), NRP-1 (neuropilin-1), PS (phosphatidylserine), RBD (receptor binding domain), SARS-CoV (severe acute respiratory syndrome coronavirus), SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), TIM-1 (T-cell immunoglobulin and mucin domain 1), TMPRSS2 (transmembrane protease, serine 2)
      Coronaviruses represent a diverse family of enveloped, positive-sense, RNA viruses infecting birds and mammals, which have become of increasing concern following three recent zoonotic transmissions of highly pathogenic human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Divided into four genera, the alpha- and beta-coronaviruses mainly circulate in bat and rodent reservoirs, whereas the gamma- and delta-coronaviruses have birds as their main reservoir species (
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      Since the identification of SARS-CoV-2 as the causative agent of COVID-19 in December of 2019 (
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      ). In particular, the mechanisms of SARS-CoV-2 entry were probed to better define the requirements for SARS-CoV-2 transmission and pathogenesis, as well as vaccine development. The SARS-CoV-2 receptor was quickly identified as angiotensin converting enzyme 2 (ACE2) (
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      ), and subsequent studies have revealed other key determinants of SARS-CoV-2 entry. Factors influencing viral entry can serve as key determinants of virus host range, tissue tropism, and pathogenicity and may be targets for therapeutics and vaccine development. Here we review the recent findings related to SARS-CoV-2 entry and place them in the context of prior coronavirus research as well as identify areas requiring further investigation.

      Receptor binding: What is unique about SARS-CoV-2 spike?

      The coronavirus spike (S) protein, one of four structural proteins, E, M, N, and S, mediates receptor binding and fusion of virus particles with target cells. The S protein is a typical type I fusion protein and functions as a trimer with each monomer divided into two subunits: S1, which mediates receptor binding, and S2, which contains the transmembrane domain and mediates fusion with the host cell membrane. Of interest, distinct from SARS-CoV yet similar to MERS-CoV, the SARS-CoV-2 S protein contains a furin proteolytic cleavage site at the S1/S2 junction, which aids in efficient viral entry, spread, and pathogenesis (
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      ). The S proteins of various coronaviruses interact with a broad range of receptors including ACE2, dipeptidyl peptidase 4, aminopeptidase-N, and sialic acid moieties to facilitate the entry process. This diversity of receptor utilization may help explain the strong potential for zoonotic transmission of coronaviruses to humans.
      Despite using the same receptor, ACE2, for binding and entry, the dynamics and receptor binding properties of SARS-CoV-2 S are somewhat different from that of SARS-CoV. Early observations indicated that the receptor-binding domain (RBD) of SARS-CoV-2 S exhibited higher affinity for ACE2 than the RBD of SARS-CoV S (
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      ), and cryo-EM structures of SARS-CoV and SARS-CoV-2 RBD in complex with ACE2 demonstrated more extensive interactions between SARS-CoV-2 RBD and ACE2 (
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      ). However, subsequent reports have suggested that full-length SARS-CoV-2 S exhibits a similar or weaker affinity for ACE2 compared with SARS-CoV S (
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      ). One possible explanation for this discrepancy is that the RBD for SARS-CoV-2 S may be less accessible to ACE2. Prior structural analyses of SARS-CoV and MERS-CoV identified an equilibrium in S proteins between an RBD-exposed conformation (open) and RBD-buried conformation (closed) (
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      ). More recently, cryo-EM-resolved SARS-CoV-2 S structures have detected a greater proportion of RBD in a closed conformation (
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      ,
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      ). This may indicate that SARS-CoV-2 S spends more time in the closed conformation than SARS-CoV S, potentially causing SARS-CoV-2 S to have a comparable affinity to ACE2 as SARS-CoV S despite the higher affinity for ACE2 of SARS-CoV-2 RBD. This may also imply an immune evasion strategy of SARS-CoV-2, where the RBD is hidden in a closed conformation to prevent the development of neutralizing antibody responses against the RBD of SARS-CoV-2 S. Further study of the triggers for a closed to open S conformational change are required to better understand how SARS-CoV-2 balances immune evasion strategies with efficient entry.

      Entry pathway and protease utilization: From plasma membrane to endosome

      As most class I viral fusion proteins, processing of the coronavirus S protein by host proteases is required for coronavirus entry. This priming process involves a cleavage at the S1/S2 boundary of the precursor S, a cleavage at the S2’ site of the S protein, or both, which results in a dissociation of the S1 subunit from S2 thus allowing S2 subunit–mediated membrane fusion between the viral envelope and the cell membrane (
      • Millet J.K.
      • Whittaker G.R.
      Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis.
      ) (Figs. 1 and 2). Previous reports have demonstrated that SARS-CoV can be primed at the S2’ site by cell membrane–associated transmembrane protease serine 2/4 (TMPRSS2/4) (
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      ). Similarly, recent evidence utilizing SARS-CoV-2 spike-pseudotyped viruses, or viral particles from commonly studied viruses bearing the SARS-CoV-2 S protein, has shown that SARS-CoV-2 can be activated via the same set of cell surface and endosomal proteases. Indeed, pharmacological inhibition of TMPRSS2 or CatB/L has been shown to reduce SARS-CoV-2 S-pseudotyped vesicular stomatitis virus or lentivirus entry (
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      ). Moreover, inhibition of TMPRSS2 with camostat mesylate is being pursued as a potential COVID-19 therapy (
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      ,
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      ). This furin cleavage site allows for proteolytic processing of SARS-CoV-2 S in the virus producer cell, typically in the trans-Golgi complex, rather than during entry into target cells. Mutation of the furin cleavage site drastically reduces the infectivity of SARS-CoV-2 S pseudotyped virus (
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      ), and virus produced from cells treated with a furin inhibitor also demonstrate a greater sensitivity to TMPRSS2 or CatB/L inhibitors for infection in HeLa cells overexpressing ACE2 (
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      ). This suggests that the presence of the furin cleavage site at the S1/S2 boundary in SARS-CoV-2 S likely reduces reliance on target cell proteases. This is unusual as cleavage at the S2’ site has been thought to be the major requirement for SARS-CoV entry (
      • Millet J.K.
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      Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis.
      ). Of interest, infectious SARS-CoV-2 possessing a furin cleavage site deletion exhibits enhanced replication in Vero-E6 cells but reduced replication in the lung epithelia-derived Calu-3 cell line (
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      ). This may indicate a need for the furin cleavage site for replication and pathogenesis in the lungs that is not necessarily maintained in all cell culture systems. It is thus not surprising that the furin cleavage site is deleted in cells lacking expression of TMPRSS2, such as Vero-E6 cells, when SARS-CoV-2 is serially passaged (
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      ). Further investigation is needed to determine the relative importance of these proteases and cleavage events in determining SARS-CoV-2 entry, pathogenesis, tropism, and host range.
      Figure thumbnail gr1
      Figure 1SARS-CoV-2 Spike contains a furin cleavage motif at S1/S2 cleavage site. Top, schematic of the SARS-CoV-2 Spike protein with S1 subunit, S2 subunit, receptor binding domain (RBD), fusion peptide, transmembrane domain, S1/S2 cleavage site, and S2’ cleavage site indicated. Bottom, alignment of SARS-CoV-2 S1/S2 and S2’ cleavage sites with corresponding regions of S protein from related bat coronaviruses (BaCoV) and other human coronaviruses. The phylogenetic tree indicates the relatedness of full-length S proteins. The RXXR furin cleavage motif at the S1/S2 site is indicated for SARS-CoV-2 and is present in MERS-CoV, HCoV-OC43, and HCoV-HKU1. The site of cleavage is indicated with an arrowhead. Sequence IDs are indicated next to the virus names and correspond to NCBI accession numbers or GISAID accession numbers. Alignment and phylogenetic tree were produced using full-length S protein sequence alignment with ClustalOmega (
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      Figure thumbnail gr2
      Figure 2SARS-CoV-2 attachment cofactors can enhance virus entry via the endosomal entry route and the plasma membrane entry route. Binding of virions to the representative attachment cofactors can facilitate SARS-CoV-2 S binding to ACE2. Then subsequent cleavage by cell surface TMPRSS2 can lead to cell membrane fusion, or endocytosis of SARS-CoV-2 allows for cathepsin B/L processing of SARS-CoV-2 S and subsequent fusion both in an ACE2-dependent manner. Whether or not TMPRSS2 processing could influence endosomal entry is currently unknown.
      The flexible protease usage of SARS-CoV-2 highlights the inherent plasticity of the virus in entry pathway usage (Fig. 2). SARS-CoV-2, SARS-CoV, and MERS-CoV are all capable of using both endosomal and plasma membrane entry routes (
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      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
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      • Gallagher T.
      Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism.
      ). TMPRSS2 appears to be one of the major proteases for priming S for entry via the plasma membrane, whereas CatB/L performs the priming function during entry through the endosome. Supporting this notion, endosomal acidification inhibitors have been shown to efficiently block SARS-CoV and SARS-CoV-2 entry by preventing the activation of CatB/L (
      • Hoffmann M.
      • Kleine-Weber H.
      • Schroeder S.
      • Krüger N.
      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.-H.
      • Nitsche A.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ,
      • Wang H.
      • Yang P.
      • Liu K.
      • Guo F.
      • Zhang Y.
      • Zhang G.
      • Jiang C.
      SARS coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway.
      ). However, low pH per se does not appear to act directly as a trigger for SARS-CoV-2 entry (
      • Zhou T.
      • Tsybovsky Y.
      • Gorman J.
      • Rapp M.
      • Cerutti G.
      • Chuang G.-Y.
      • Katsamba P.S.
      • Sampson J.M.
      • Schön A.
      • Bimela J.
      Cryo-EM structures of SARS-CoV-2 spike without and with ACE2 reveal a pH-dependent switch to mediate endosomal positioning of receptor-binding domains.
      ). It should be noted, however, that recent reports suggest that pH 5.5 to 6.0 can stabilize the SARS-CoV-2 S protein and cause a shift toward a more open conformation, potentially facilitating viral membrane fusion in the endosome (
      • Zhou T.
      • Tsybovsky Y.
      • Gorman J.
      • Rapp M.
      • Cerutti G.
      • Chuang G.-Y.
      • Katsamba P.S.
      • Sampson J.M.
      • Schön A.
      • Bimela J.
      Cryo-EM structures of SARS-CoV-2 spike without and with ACE2 reveal a pH-dependent switch to mediate endosomal positioning of receptor-binding domains.
      ). This stability and opening of the S protein at endosomal pH may suggest a preference for an endosomal entry route. However, cell culture studies have demonstrated that TMPRSS2 inhibitors more drastically reduce SARS-CoV-2 entry compared with CatB/L inhibitors in physiologically relevant cell types such as Calu-3 cells (
      • Hoffmann M.
      • Kleine-Weber H.
      • Schroeder S.
      • Krüger N.
      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.-H.
      • Nitsche A.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ). In addition, it has been shown for MERS-CoV that furin cleavage in producer cells leads to a preference for subsequent processing by TMPRSS2 (
      • Park J.-E.
      • Li K.
      • Barlan A.
      • Fehr A.R.
      • Perlman S.
      • McCray P.B.
      • Gallagher T.
      Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism.
      ,
      • Kleine-Weber H.
      • Elzayat M.T.
      • Hoffmann M.
      • Pöhlmann S.
      Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein.
      ). Prior reports suggest that other human coronaviruses, such as HCoV-OC43 and HCoV-HKU1, exhibit a preference for a cell membrane entry route and only acquire an ability to utilize CatB/L upon passaging in HBTE-ALI cell culture while accumulating a furin cleavage site mutation (
      • Shirato K.
      • Kawase M.
      • Matsuyama S.
      Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry.
      ). This is noteworthy as the passaging of SARS-CoV-2 in Vero-E6 cells has led to the problematic accumulation of furin cleavage site deletions or mutations (
      • Davidson A.D.
      • Williamson M.K.
      • Lewis S.
      • Shoemark D.
      • Carroll M.W.
      • Heesom K.J.
      • Zambon M.
      • Ellis J.
      • Lewis P.A.
      • Hiscox J.A.
      Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
      ,
      • Ogando N.S.
      • Dalebout T.J.
      • Zevenhoven-Dobbe J.C.
      • Limpens R.W.
      • van der Meer Y.
      • Caly L.
      • Druce J.
      • de Vries J.J.
      • Kikkert M.
      • Barcena M.
      • Sidorov I.
      • Snijder E.J.
      SARS-coronavirus-2 replication in Vero E6 cells: Replication kinetics, rapid adaptation and cytopathology.
      ,
      • Liu Z.
      • Zheng H.
      • Lin H.
      • Li M.
      • Yuan R.
      • Peng J.
      • Xiong Q.
      • Sun J.
      • Li B.
      • Wu J.
      • Yi L.
      • Peng X.
      • Zhang H.
      • Zhang W.
      • Hulswit R.J.
      • et al.
      Identification of common deletions in the spike protein of severe acute respiratory syndrome coronavirus 2.
      ), perhaps suggesting a shift toward an endosomal entry route in Vero-E6 culture. Hence, the flexibility in protease usage and entry route appears to be a consistent strategy used by coronaviruses, despite in some cases acting in a cell type–dependent manner. More investigation is needed to determine the physiological relevance of each entry pathway to SARS-CoV-2 spread and pathogenesis in vivo.

      Cellular entry modulators: Receptor versus cofactor

      Spike/receptor interactions and protease processing of the spike are not the only factors determining coronavirus entry. A number of cellular proteins, referred to as cofactors here, have been identified to enhance the attachment and entry of coronavirus particles into target cells. Such entry cofactors are sometimes referred to as viral receptors in the literature, which has caused much confusion. Although defining a viral receptor can be challenging, it should include at least a demonstration of the direct interaction with viral protein of interest in vitro and the induction of conformational changes in the viral protein (
      • Evans J.P.
      • Liu S.-L.
      Multifaceted roles of TIM-family proteins in virus–host interactions.
      ). Caution should be exercised in distinguishing a bona fide receptor from a cellular cofactor. Currently, ACE2 is the only primary receptor identified for SARS-CoV-2, the highly related SARS-CoV, as well as distantly related HCoV-NL63. Here we outline important coronavirus entry cofactors and ongoing work to determine their role, if any, in SARS-CoV-2 entry (Table 1).
      Table 1Host factors affecting human coronavirus entry
      VirusReceptorCofactorRestriction factor
      SARS-CoV-2ACE2 (
      • Wu F.
      • Zhao S.
      • Yu B.
      • Chen Y.-M.
      • Wang W.
      • Song Z.-G.
      • Hu Y.
      • Tao Z.-W.
      • Tian J.-H.
      • Pei Y.-Y.
      A new coronavirus associated with human respiratory disease in China.
      ,
      • Zhou P.
      • Yang X.-L.
      • Wang X.-G.
      • Hu B.
      • Zhang L.
      • Zhang W.
      • Si H.-R.
      • Zhu Y.
      • Li B.
      • Huang C.-L.
      A pneumonia outbreak associated with a new coronavirus of probable bat origin.
      ,
      • Wrapp D.
      • Wang N.
      • Corbett K.S.
      • Goldsmith J.A.
      • Hsieh C.-L.
      • Abiona O.
      • Graham B.S.
      • McLellan J.S.
      Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.
      )
      TMPRSS2 (
      • Shang J.
      • Wan Y.
      • Luo C.
      • Ye G.
      • Geng Q.
      • Auerbach A.
      • Li F.
      Cell entry mechanisms of SARS-CoV-2.
      ,
      • Hoffmann M.
      • Kleine-Weber H.
      • Schroeder S.
      • Krüger N.
      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.-H.
      • Nitsche A.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      )
      IFITMs (
      • Bozzo C.P.
      • Nchioua R.
      • Volcic M.
      • Wettstein L.
      • Weil T.
      • Krüger J.
      • Heller S.
      • Conzelmann C.
      • Mueller J.A.
      • Gross R.
      IFITM proteins promote SARS-CoV-2 infection in human lung cells.
      ,
      • Shi G.
      • Kenney A.D.
      • Kudryashova E.
      • Zani A.
      • Zhang L.
      • Lai K.K.
      • Hall-Stoodley L.
      • Robinson R.T.
      • Kudryashov D.S.
      • Compton A.A.
      Opposing activities of IFITM proteins in SARS-CoV-2 infection.
      ,
      • Buchrieser J.
      • Dufloo J.
      • Hubert M.
      • Monel B.
      • Planas D.
      • Rajah M.M.
      • Planchais C.
      • Porrot F.
      • Guivel-Benhassine F.
      • Van der Werf S.
      Syncytia formation by SARS-CoV-2-infected cells.
      )
      CatB/L (
      • Shang J.
      • Wan Y.
      • Luo C.
      • Ye G.
      • Geng Q.
      • Auerbach A.
      • Li F.
      Cell entry mechanisms of SARS-CoV-2.
      ,
      • Hoffmann M.
      • Kleine-Weber H.
      • Schroeder S.
      • Krüger N.
      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.-H.
      • Nitsche A.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      )
      NRP-1 (
      • Cantuti-Castelvetri L.
      • Ojha R.
      • Pedro L.D.
      • Djannatian M.
      • Franz J.
      • Kuivanen S.
      • van der Meer F.
      • Kallio K.
      • Kaya T.
      • Anastasina M.
      Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.
      ,
      • Daly J.L.
      • Simonetti B.
      • Klein K.
      • Chen K.-E.
      • Williamson M.K.
      • Anton-Plagaro C.
      • Shoemark D.K.
      • Simon-Gracia L.
      • Bauer M.
      • Hollandi R.
      • Greber U.F.
      • Horvath P.
      • Sessions R.B.
      • Helenius A.
      • Hiscox J.A.
      • et al.
      Neuropilin-1 is a host factor for SARS-CoV-2 infection.
      )
      CD147 (
      • Wang K.
      • Chen W.
      • Zhang Z.
      • Deng Y.
      • Lian J.-Q.
      • Du P.
      • Wei D.
      • Zhang Y.
      • Sun X.-X.
      • Gong L.
      CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells.
      )
      Ly6E (
      • Zhao X.
      • Zheng S.
      • Chen D.
      • Zheng M.
      • Li X.
      • Li G.
      • Lin H.
      • Chang J.
      • Zeng H.
      • Guo J.-T.
      LY6E restricts entry of human coronaviruses, including currently pandemic SARS-CoV-2.
      ,
      • Pfaender S.
      • Mar K.B.
      • Michailidis E.
      • Kratzel A.
      • Boys I.N.
      • V’kovski P.
      • Fan W.
      • Kelly J.N.
      • Hirt D.
      • Ebert N.
      • Stalder H.
      • Kleine-Weber H.
      • Hoffmann M.
      • Hoffmann H.-H.
      • Saeed M.
      • et al.
      LY6E impairs coronavirus fusion and confers immune control of viral disease.
      )
      Axl (
      • Wang S.
      • Qiu Z.
      • Hou Y.
      • Deng X.
      • Xu W.
      • Zheng T.
      • Wu P.
      • Xie S.
      • Bian W.
      • Zhang C.
      AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells.
      )
      TIM-1
      Designation is based solely on data that has not yet undergone peer review.
      (
      • Ichimura T.
      • Mori Y.
      • Aschauer P.
      • Das K.M.P.
      • Padera R.F.
      • Weins A.
      • Nasr M.L.
      • Bonventre J.V.
      KIM-1/TIM-1 is a receptor for SARS-CoV-2 in lung and kidney.
      )
      HSPGs (
      • Clausen T.M.
      • Sandoval D.R.
      • Spliid C.B.
      • Pihl J.
      • Perrett H.R.
      • Painter C.D.
      • Narayanan A.
      • Majowicz S.A.
      • Kwong E.M.
      • McVicar R.N.
      SARS-CoV-2 infection depends on cellular heparan sulfate and ACE2.
      ,
      • Zhang Q.
      • Chen C.Z.
      • Swaroop M.
      • Xu M.
      • Wang L.
      • Lee J.
      • Wang A.Q.
      • Pradhan M.
      • Hagen N.
      • Chen L.
      Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro.
      ,
      • Kim S.Y.
      • Jin W.
      • Sood A.
      • Montgomery D.W.
      • Grant O.C.
      • Fuster M.M.
      • Fu L.
      • Dordick J.S.
      • Woods R.J.
      • Zhang F.
      Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions.
      ,
      • Kwon P.S.
      • Oh H.
      • Kwon S.-J.
      • Jin W.
      • Zhang F.
      • Fraser K.
      • Hong J.J.
      • Linhardt R.J.
      • Dordick J.S.
      Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro.
      )
      C-type lectin (
      • Thepaut M.
      • Luczkowiak J.
      • Vives C.
      • Labiod N.
      • Bally I.
      • Lasala F.
      • Grimoire Y.
      • Fenel D.
      • Sattin S.
      • Thielens N.
      • Schoehn G.
      • Bernardi A.
      • Delgado R.
      • Fieschi F.
      DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist.
      ,
      • Amraei R.
      • Napoleon M.
      • Yin W.
      • Berrigan J.
      • Suder E.
      • Zhao G.
      • Olejnik J.
      • Gummuluru S.
      • Muhlberger E.
      • Chitalia V.
      CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2 and are differentially expressed in lung and kidney epithelial and endothelial cells.
      )
      SARS-CoVACE2 (
      • Li W.
      • Moore M.J.
      • Vasilieva N.
      • Sui J.
      • Wong S.K.
      • Berne M.A.
      • Somasundaran M.
      • Sullivan J.L.
      • Luzuriaga K.
      • Greenough T.C.
      Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.
      )
      TMPRSS2 (
      • Bertram S.
      • Glowacka I.
      • Müller M.A.
      • Lavender H.
      • Gnirss K.
      • Nehlmeier I.
      • Niemeyer D.
      • He Y.
      • Simmons G.
      • Drosten C.
      Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease.
      ,
      • Matsuyama S.
      • Nagata N.
      • Shirato K.
      • Kawase M.
      • Takeda M.
      • Taguchi F.
      Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2.
      )
      IFITMs (
      • Huang I.-C.
      • Bailey C.C.
      • Weyer J.L.
      • Radoshitzky S.R.
      • Becker M.M.
      • Chiang J.J.
      • Brass A.L.
      • Ahmed A.A.
      • Chi X.
      • Dong L.
      Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.
      )
      CatB/L (
      • Bertram S.
      • Glowacka I.
      • Müller M.A.
      • Lavender H.
      • Gnirss K.
      • Nehlmeier I.
      • Niemeyer D.
      • He Y.
      • Simmons G.
      • Drosten C.
      Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease.
      ,
      • Simmons G.
      • Gosalia D.N.
      • Rennekamp A.J.
      • Reeves J.D.
      • Diamond S.L.
      • Bates P.
      Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry.
      )
      CypA/B (
      • Chen Z.
      • Mi L.
      • Xu J.
      • Yu J.
      • Wang X.
      • Jiang J.
      • Xing J.
      • Shang P.
      • Qian A.
      • Li Y.
      Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus.
      ,
      • de Wilde A.H.
      • Zevenhoven-Dobbe J.C.
      • van der Meer Y.
      • Thiel V.
      • Narayanan K.
      • Makino S.
      • Snijder E.J.
      • van Hemert M.J.
      Cyclosporin A inhibits the replication of diverse coronaviruses.
      ,
      • Pfefferle S.
      • Schöpf J.
      • Kögl M.
      • Friedel C.C.
      • Müller M.A.
      • Carbajo-Lozoya J.
      • Stellberger T.
      • von Dall’Armi E.
      • Herzog P.
      • Kallies S.
      The SARS-coronavirus-host interactome: Identification of cyclophilins as target for pan-coronavirus inhibitors.
      )
      Ly6E (
      • Pfaender S.
      • Mar K.B.
      • Michailidis E.
      • Kratzel A.
      • Boys I.N.
      • V’kovski P.
      • Fan W.
      • Kelly J.N.
      • Hirt D.
      • Ebert N.
      • Stalder H.
      • Kleine-Weber H.
      • Hoffmann M.
      • Hoffmann H.-H.
      • Saeed M.
      • et al.
      LY6E impairs coronavirus fusion and confers immune control of viral disease.
      )
      HSPGs (
      • Lang J.
      • Yang N.
      • Deng J.
      • Liu K.
      • Yang P.
      • Zhang G.
      • Jiang C.
      Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans.
      )
      C-type lectin (
      • Marzi A.
      • Gramberg T.
      • Simmons G.
      • Möller P.
      • Rennekamp A.J.
      • Krumbiegel M.
      • Geier M.
      • Eisemann J.
      • Turza N.
      • Saunier B.
      DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus.
      ,
      • Yang Z.-Y.
      • Huang Y.
      • Ganesh L.
      • Leung K.
      • Kong W.-P.
      • Schwartz O.
      • Subbarao K.
      • Nabel G.J.
      pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN.
      ,
      • Han D.P.
      • Lohani M.
      • Cho M.W.
      Specific asparagine-linked glycosylation sites are critical for DC-SIGN-and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry.
      )
      MERS-CoVDPP4 (
      • Raj V.S.
      • Mou H.
      • Smits S.L.
      • Dekkers D.H.
      • Müller M.A.
      • Dijkman R.
      • Muth D.
      • Demmers J.A.
      • Zaki A.
      • Fouchier R.A.
      Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.
      )
      TMPRSS2 (
      • Park J.-E.
      • Li K.
      • Barlan A.
      • Fehr A.R.
      • Perlman S.
      • McCray P.B.
      • Gallagher T.
      Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism.
      ,
      • Kleine-Weber H.
      • Elzayat M.T.
      • Hoffmann M.
      • Pöhlmann S.
      Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein.
      ,
      • Shirato K.
      • Kawase M.
      • Matsuyama S.
      Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2.
      )
      IFITMs (
      • Wrensch F.
      • Winkler M.
      • Pöhlmann S.
      IFITM proteins inhibit entry driven by the MERS-coronavirus spike protein: Evidence for cholesterol-independent mechanisms.
      )
      CatB/L (
      • Qian Z.
      • Dominguez S.R.
      • Holmes K.V.
      Role of the spike glycoprotein of human Middle East respiratory syndrome coronavirus (MERS-CoV) in virus entry and syncytia formation.
      )
      CypA/B (
      • de Wilde A.H.
      • Raj V.S.
      • Oudshoorn D.
      • Bestebroer T.M.
      • van Nieuwkoop S.
      • Limpens R.W.
      • Posthuma C.C.
      • van der Meer Y.
      • Bárcena M.
      • Haagmans B.L.
      MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment.
      )
      Ly6E (
      • Zhao X.
      • Zheng S.
      • Chen D.
      • Zheng M.
      • Li X.
      • Li G.
      • Lin H.
      • Chang J.
      • Zeng H.
      • Guo J.-T.
      LY6E restricts entry of human coronaviruses, including currently pandemic SARS-CoV-2.
      ,
      • Pfaender S.
      • Mar K.B.
      • Michailidis E.
      • Kratzel A.
      • Boys I.N.
      • V’kovski P.
      • Fan W.
      • Kelly J.N.
      • Hirt D.
      • Ebert N.
      • Stalder H.
      • Kleine-Weber H.
      • Hoffmann M.
      • Hoffmann H.-H.
      • Saeed M.
      • et al.
      LY6E impairs coronavirus fusion and confers immune control of viral disease.
      )
      Sialic Acid (
      • Li W.
      • Hulswit R.J.
      • Widjaja I.
      • Raj V.S.
      • McBride R.
      • Peng W.
      • Widagdo W.
      • Tortorici M.A.
      • Van Dieren B.
      • Lang Y.
      Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.
      ,
      • Park Y.-J.
      • Walls A.C.
      • Wang Z.
      • Sauer M.M.
      • Li W.
      • Tortorici M.A.
      • Bosch B.-J.
      • DiMaio F.
      • Veesler D.
      Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors.
      )
      HCoV-NL63ACE2 (
      • Hofmann H.
      • Pyrc K.
      • Van Der Hoek L.
      • Geier M.
      • Berkhout B.
      • Pöhlmann S.
      Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry.
      )
      CypA/B (
      • Carbajo-Lozoya J.
      • Ma-Lauer Y.
      • Malešević M.
      • Theuerkorn M.
      • Kahlert V.
      • Prell E.
      • Von Brunn B.
      • Muth D.
      • Baumert T.F.
      • Drosten C.
      Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including Alisporivir.
      )
      IFITMs (
      • Zhao X.
      • Sehgal M.
      • Hou Z.
      • Cheng J.
      • Shu S.
      • Wu S.
      • Guo F.
      • Le Marchand S.J.
      • Lin H.
      • Chang J.
      Identification of residues controlling restriction versus enhancing activities of IFITM proteins on entry of human coronaviruses.
      )
      HSPGs (
      • Milewska A.
      • Zarebski M.
      • Nowak P.
      • Stozek K.
      • Potempa J.
      • Pyrc K.
      Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells.
      )
      Ly6E (
      • Zhao X.
      • Zheng S.
      • Chen D.
      • Zheng M.
      • Li X.
      • Li G.
      • Lin H.
      • Chang J.
      • Zeng H.
      • Guo J.-T.
      LY6E restricts entry of human coronaviruses, including currently pandemic SARS-CoV-2.
      )
      C-type lectin (
      • Hofmann H.
      • Simmons G.
      • Rennekamp A.J.
      • Chaipan C.
      • Gramberg T.
      • Heck E.
      • Geier M.
      • Wegele A.
      • Marzi A.
      • Bates P.
      Highly conserved regions within the spike proteins of human coronaviruses 229E and NL63 determine recognition of their respective cellular receptors.
      )
      HCoV-OC439-O-Ac-Sia (
      • Vlasak R.
      • Luytjes W.
      • Spaan W.
      • Palese P.
      Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses.
      )
      TMPRSS2 (
      • Shirato K.
      • Kawase M.
      • Matsuyama S.
      Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry.
      )
      Ly6E (
      • Zhao X.
      • Zheng S.
      • Chen D.
      • Zheng M.
      • Li X.
      • Li G.
      • Lin H.
      • Chang J.
      • Zeng H.
      • Guo J.-T.
      LY6E restricts entry of human coronaviruses, including currently pandemic SARS-CoV-2.
      ,
      • Pfaender S.
      • Mar K.B.
      • Michailidis E.
      • Kratzel A.
      • Boys I.N.
      • V’kovski P.
      • Fan W.
      • Kelly J.N.
      • Hirt D.
      • Ebert N.
      • Stalder H.
      • Kleine-Weber H.
      • Hoffmann M.
      • Hoffmann H.-H.
      • Saeed M.
      • et al.
      LY6E impairs coronavirus fusion and confers immune control of viral disease.
      )
      CatB/L (
      • Shirato K.
      • Kawase M.
      • Matsuyama S.
      Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry.
      )
      HSPGs (
      • Szczepanski A.
      • Owczarek K.
      • Bzowska M.
      • Gula K.
      • Drebot I.
      • Ochman M.
      • Maksym B.
      • Rajfur Z.
      • Mitchell J.A.
      • Pyrc K.
      Canine respiratory coronavirus, bovine coronavirus, and human coronavirus OC43: Receptors and attachment factors.
      )
      HLA-1 (
      • Collins A.R.
      Human coronavirus OC43 interacts with major histocompatibility complex class I molecules at the cell surface to establish infection.
      )
      IFITMs (
      • Zhao X.
      • Guo F.
      • Liu F.
      • Cuconati A.
      • Chang J.
      • Block T.M.
      • Guo J.-T.
      Interferon induction of IFITM proteins promotes infection by human coronavirus OC43.
      )
      HCoV-HKU19-O-Ac-Sia (
      • Huang X.
      • Dong W.
      • Milewska A.
      • Golda A.
      • Qi Y.
      • Zhu Q.K.
      • Marasco W.A.
      • Baric R.S.
      • Sims A.C.
      • Pyrc K.
      Human coronavirus HKU1 spike protein uses O-acetylated sialic acid as an attachment receptor determinant and employs hemagglutinin-esterase protein as a receptor-destroying enzyme.
      )
      TMPRSS2 (
      • Shirato K.
      • Kawase M.
      • Matsuyama S.
      Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry.
      )
      CatB/L (
      • Shirato K.
      • Kawase M.
      • Matsuyama S.
      Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry.
      )
      HLA-C (
      • Chan C.M.
      • Lau S.K.
      • Woo P.C.
      • Tse H.
      • Zheng B.-J.
      • Chen L.
      • Huang J.-D.
      • Yuen K.-Y.
      Identification of major histocompatibility complex class IC molecule as an attachment factor that facilitates coronavirus HKU1 spike-mediated infection.
      )
      HCoV-229EhAPN (
      • Yeager C.L.
      • Ashmun R.A.
      • Williams R.K.
      • Cardellichio C.B.
      • Shapiro L.H.
      • Look A.T.
      • Holmes K.V.
      Human aminopeptidase N is a receptor for human coronavirus 229E.
      )
      TMPRSS2 (
      • Bertram S.
      • Dijkman R.
      • Habjan M.
      • Heurich A.
      • Gierer S.
      • Glowacka I.
      • Welsch K.
      • Winkler M.
      • Schneider H.
      • Hofmann-Winkler H.
      TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium.
      )
      IFITMs (
      • Zhao X.
      • Sehgal M.
      • Hou Z.
      • Cheng J.
      • Shu S.
      • Wu S.
      • Guo F.
      • Le Marchand S.J.
      • Lin H.
      • Chang J.
      Identification of residues controlling restriction versus enhancing activities of IFITM proteins on entry of human coronaviruses.
      )
      Cat B/L (
      • Kawase M.
      • Shirato K.
      • Matsuyama S.
      • Taguchi F.
      Protease-mediated entry via the endosome of human coronavirus 229E.
      )
      CypA/B (
      • Von Brunn A.
      • Ciesek S.
      • Von Brunn B.
      • Carbajo-Lozoya J.
      Genetic deficiency and polymorphisms of cyclophilin A reveal its essential role for human coronavirus 229E replication.
      ,
      • de Wilde A.H.
      • Zevenhoven-Dobbe J.C.
      • van der Meer Y.
      • Thiel V.
      • Narayanan K.
      • Makino S.
      • Snijder E.J.
      • van Hemert M.J.
      Cyclosporin A inhibits the replication of diverse coronaviruses.
      )
      Ly6E (
      • Pfaender S.
      • Mar K.B.
      • Michailidis E.
      • Kratzel A.
      • Boys I.N.
      • V’kovski P.
      • Fan W.
      • Kelly J.N.
      • Hirt D.
      • Ebert N.
      • Stalder H.
      • Kleine-Weber H.
      • Hoffmann M.
      • Hoffmann H.-H.
      • Saeed M.
      • et al.
      LY6E impairs coronavirus fusion and confers immune control of viral disease.
      )
      C-type lectin (
      • Jeffers S.A.
      • Hemmila E.M.
      • Holmes K.V.
      Human coronavirus 229E can use CD209L (L-SIGN) to enter cells.
      )
      Abbreviations: 9-O-Ac-Sia, 9-O-acetylated sialic acid; DPP4, dipeptidyl peptidase 4; hAPN, human aminopeptidase-N; TMPRSS2, Transmembrane protease, serine 2.
      a Designation is based solely on data that has not yet undergone peer review.
      Neuropilin-1 (NRP-1) has recently been identified as an entry cofactor for SARS-CoV-2 (
      • Cantuti-Castelvetri L.
      • Ojha R.
      • Pedro L.D.
      • Djannatian M.
      • Franz J.
      • Kuivanen S.
      • van der Meer F.
      • Kallio K.
      • Kaya T.
      • Anastasina M.
      Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.
      ,
      • Daly J.L.
      • Simonetti B.
      • Klein K.
      • Chen K.-E.
      • Williamson M.K.
      • Anton-Plagaro C.
      • Shoemark D.K.
      • Simon-Gracia L.
      • Bauer M.
      • Hollandi R.
      • Greber U.F.
      • Horvath P.
      • Sessions R.B.
      • Helenius A.
      • Hiscox J.A.
      • et al.
      Neuropilin-1 is a host factor for SARS-CoV-2 infection.
      ). It is intriguing that NRP-1 was shown to bind furin cleavage products containing an R/KXXR/K motif (
      • Teesalu T.
      • Sugahara K.N.
      • Kotamraju V.R.
      • Ruoslahti E.
      C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration.
      ). Given the presence of such a furin cleavage site in the S of SARS-CoV-2, two groups recently investigated the role of NRP-1 in SARS-CoV-2 S-mediated entry (
      • Cantuti-Castelvetri L.
      • Ojha R.
      • Pedro L.D.
      • Djannatian M.
      • Franz J.
      • Kuivanen S.
      • van der Meer F.
      • Kallio K.
      • Kaya T.
      • Anastasina M.
      Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.
      ,
      • Daly J.L.
      • Simonetti B.
      • Klein K.
      • Chen K.-E.
      • Williamson M.K.
      • Anton-Plagaro C.
      • Shoemark D.K.
      • Simon-Gracia L.
      • Bauer M.
      • Hollandi R.
      • Greber U.F.
      • Horvath P.
      • Sessions R.B.
      • Helenius A.
      • Hiscox J.A.
      • et al.
      Neuropilin-1 is a host factor for SARS-CoV-2 infection.
      ). NRP-1 knockdown in Caco-2 cells, or knockout in HeLa cells stably expressing ACE2, reduces infectious SARS-CoV-2 replication (
      • Daly J.L.
      • Simonetti B.
      • Klein K.
      • Chen K.-E.
      • Williamson M.K.
      • Anton-Plagaro C.
      • Shoemark D.K.
      • Simon-Gracia L.
      • Bauer M.
      • Hollandi R.
      • Greber U.F.
      • Horvath P.
      • Sessions R.B.
      • Helenius A.
      • Hiscox J.A.
      • et al.
      Neuropilin-1 is a host factor for SARS-CoV-2 infection.
      ). In addition, overexpression of NRP-1 in Caco-2 cells, which endogenously express ACE2, enhanced SARS-CoV-2 S pseudotyped virus entry (
      • Cantuti-Castelvetri L.
      • Ojha R.
      • Pedro L.D.
      • Djannatian M.
      • Franz J.
      • Kuivanen S.
      • van der Meer F.
      • Kallio K.
      • Kaya T.
      • Anastasina M.
      Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.
      ), indicating a role for NRP-1 in SARS-CoV-2 entry. However, overexpression of NRP-1 in HEK 293T cells only enhances SARS-CoV-2 pseudotyped virus infection when both ACE2 and TMPRSS2 are coexpressed (
      • Cantuti-Castelvetri L.
      • Ojha R.
      • Pedro L.D.
      • Djannatian M.
      • Franz J.
      • Kuivanen S.
      • van der Meer F.
      • Kallio K.
      • Kaya T.
      • Anastasina M.
      Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.
      ). Hence, it appears that NRP-1 does not function as a bona fide receptor for SARS-CoV-2 as entry still requires ACE2, indicating that NRP-1 is likely serving as an entry cofactor. Of note, NRP-1 seems to directly bind SARS-CoV-2 S as shown by coimmunoprecipitation and isothermal titration calorimetry (
      • Daly J.L.
      • Simonetti B.
      • Klein K.
      • Chen K.-E.
      • Williamson M.K.
      • Anton-Plagaro C.
      • Shoemark D.K.
      • Simon-Gracia L.
      • Bauer M.
      • Hollandi R.
      • Greber U.F.
      • Horvath P.
      • Sessions R.B.
      • Helenius A.
      • Hiscox J.A.
      • et al.
      Neuropilin-1 is a host factor for SARS-CoV-2 infection.
      ), and this interaction is ablated for SARS-CoV-2 S with a furin cleavage site mutation (
      • Daly J.L.
      • Simonetti B.
      • Klein K.
      • Chen K.-E.
      • Williamson M.K.
      • Anton-Plagaro C.
      • Shoemark D.K.
      • Simon-Gracia L.
      • Bauer M.
      • Hollandi R.
      • Greber U.F.
      • Horvath P.
      • Sessions R.B.
      • Helenius A.
      • Hiscox J.A.
      • et al.
      Neuropilin-1 is a host factor for SARS-CoV-2 infection.
      ). Furthermore, SARS-CoV-2 S pseudotyped virus entry is not perturbed by NRP-1 antibody blockade for S containing a furin cleavage site mutation (
      • Cantuti-Castelvetri L.
      • Ojha R.
      • Pedro L.D.
      • Djannatian M.
      • Franz J.
      • Kuivanen S.
      • van der Meer F.
      • Kallio K.
      • Kaya T.
      • Anastasina M.
      Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.
      ). This would indicate that NRP-1 enhancement of SARS-CoV-2 entry is dependent on furin cleavage of the S protein. Indeed, expression of NRP-1 in the olfactory epithelium and airway (
      • Cantuti-Castelvetri L.
      • Ojha R.
      • Pedro L.D.
      • Djannatian M.
      • Franz J.
      • Kuivanen S.
      • van der Meer F.
      • Kallio K.
      • Kaya T.
      • Anastasina M.
      Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.
      ) appears to facilitate SARS-CoV-2 entry despite the modest ACE2 expression seen in the human airway. In addition, silver nanoparticles coated with furin–cleaved SARS-CoV-2 S mimetic peptides were also more efficiently taken up by the olfactory epithelium and central nervous system of mice than nanoparticles coated with uncleaved S mimetic peptides (
      • Cantuti-Castelvetri L.
      • Ojha R.
      • Pedro L.D.
      • Djannatian M.
      • Franz J.
      • Kuivanen S.
      • van der Meer F.
      • Kallio K.
      • Kaya T.
      • Anastasina M.
      Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.
      ). Whether or not NRP-1 functions in other tissues and is related to COVID-19 pathogenesis needs to be determined.
      Cluster of differentiation 147 (CD147) has been shown to enhance the entry of several enveloped viruses (
      • Chen Z.
      • Mi L.
      • Xu J.
      • Yu J.
      • Wang X.
      • Jiang J.
      • Xing J.
      • Shang P.
      • Qian A.
      • Li Y.
      Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus.
      ,
      • Pushkarsky T.
      • Zybarth G.
      • Dubrovsky L.
      • Yurchenko V.
      • Tang H.
      • Guo H.
      • Toole B.
      • Sherry B.
      • Bukrinsky M.
      CD147 facilitates HIV-1 infection by interacting with virus-associated cyclophilin A.
      ,
      • Vanarsdall A.L.
      • Pritchard S.R.
      • Wisner T.W.
      • Liu J.
      • Jardetzky T.S.
      • Johnson D.C.
      CD147 promotes entry of pentamer-expressing human cytomegalovirus into epithelial and endothelial cells.
      ,
      • Watanabe A.
      • Yoneda M.
      • Ikeda F.
      • Terao-Muto Y.
      • Sato H.
      • Kai C.
      CD147/EMMPRIN acts as a functional entry receptor for measles virus on epithelial cells.
      ). This is due to binding of CD147 to virion-associated cyclophilin A or B (CypA/B) (
      • Chen Z.
      • Mi L.
      • Xu J.
      • Yu J.
      • Wang X.
      • Jiang J.
      • Xing J.
      • Shang P.
      • Qian A.
      • Li Y.
      Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus.
      ,
      • Pushkarsky T.
      • Zybarth G.
      • Dubrovsky L.
      • Yurchenko V.
      • Tang H.
      • Guo H.
      • Toole B.
      • Sherry B.
      • Bukrinsky M.
      CD147 facilitates HIV-1 infection by interacting with virus-associated cyclophilin A.
      ,
      • Yurchenko V.
      • Constant S.
      • Bukrinsky M.
      Dealing with the family: CD147 interactions with cyclophilins.
      ). In fact, the replication of HCoV-229E, HCoV-NL63, mouse hepatits virus (MHV), transmissible gastroenteritis virus, infectious bronchitis virus, feline coronavirus, and SARS-CoV has been found to be dependent on CypA/B and blocked by the CypA/B inhibitor cyclosporin A (
      • Von Brunn A.
      • Ciesek S.
      • Von Brunn B.
      • Carbajo-Lozoya J.
      Genetic deficiency and polymorphisms of cyclophilin A reveal its essential role for human coronavirus 229E replication.
      ,
      • Carbajo-Lozoya J.
      • Ma-Lauer Y.
      • Malešević M.
      • Theuerkorn M.
      • Kahlert V.
      • Prell E.
      • Von Brunn B.
      • Muth D.
      • Baumert T.F.
      • Drosten C.
      Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including Alisporivir.
      ,
      • de Wilde A.H.
      • Zevenhoven-Dobbe J.C.
      • van der Meer Y.
      • Thiel V.
      • Narayanan K.
      • Makino S.
      • Snijder E.J.
      • van Hemert M.J.
      Cyclosporin A inhibits the replication of diverse coronaviruses.
      ,
      • Pfefferle S.
      • Schöpf J.
      • Kögl M.
      • Friedel C.C.
      • Müller M.A.
      • Carbajo-Lozoya J.
      • Stellberger T.
      • von Dall’Armi E.
      • Herzog P.
      • Kallies S.
      The SARS-coronavirus-host interactome: Identification of cyclophilins as target for pan-coronavirus inhibitors.
      ,
      • Tanaka Y.
      • Sato Y.
      • Sasaki T.
      Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B.
      ). Specifically, SARS-CoV N protein has been shown to interact with CypA and enhance SARS-CoV entry via CD147/CypA interactions on target cells (
      • Chen Z.
      • Mi L.
      • Xu J.
      • Yu J.
      • Wang X.
      • Jiang J.
      • Xing J.
      • Shang P.
      • Qian A.
      • Li Y.
      Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus.
      ). In addition, CD147 blockade was shown to reduce SARS-CoV-2 replication in Vero-E6 cells and direct interaction was seen between recombinant SARS-CoV-2 S and CD147 (
      • Wang K.
      • Chen W.
      • Zhang Z.
      • Deng Y.
      • Lian J.-Q.
      • Du P.
      • Wei D.
      • Zhang Y.
      • Sun X.-X.
      • Gong L.
      CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells.
      ). However, it remains to be determined if CypA/B or SARS-CoV-2 N plays a role in entry enhancement by CD147. Although CD147 has been claimed as a bona fide receptor for SARS-CoV-2 (
      • Wang K.
      • Chen W.
      • Zhang Z.
      • Deng Y.
      • Lian J.-Q.
      • Du P.
      • Wei D.
      • Zhang Y.
      • Sun X.-X.
      • Gong L.
      CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells.
      ,
      • Ulrich H.
      • Pillat M.M.
      CD147 as a target for COVID-19 treatment: Suggested effects of azithromycin and stem cell engagement.
      ), current data seem to support the notion that CD147 acts as an attachment cofactor to facilitate SARS-CoV-2 entry. It is interesting that disruption of CD147 and CypA/B enhancement of SARS-CoV-2 entry has been suggested as a viable therapeutic strategy for the treatment of COVID-19 (
      • Ulrich H.
      • Pillat M.M.
      CD147 as a target for COVID-19 treatment: Suggested effects of azithromycin and stem cell engagement.
      ,
      • Raony Í.
      • de Figueiredo C.S.
      Retinal outcomes of COVID-19: Possible role of CD147 and cytokine storm in infected patients with diabetes mellitus.
      ,
      • Liu C.
      • Zhu D.
      Cyclophilin A and CD147: Novel therapeutic targets for the treatment of COVID-19.
      ); however, the mechanism of CD147 enhancement of SARS-CoV-2 entry as well as its possible role in SARS-CoV-2 pathogenesis is currently unclear and requires investigation.
      Phosphatidylserine (PS) receptors are another key set of viral entry cofactors. These PS receptors largely act by binding to PS, a modified membrane lipid, that is incorporated into enveloped or nonenveloped virus particles to enhance their attachment to target cells (
      • Evans J.P.
      • Liu S.-L.
      Multifaceted roles of TIM-family proteins in virus–host interactions.
      ,
      • Moller-Tank S.
      • Maury W.
      Phosphatidylserine receptors: Enhancers of enveloped virus entry and infection.
      ). This process of PS-mediated enhancement of virus attachment is known as apoptotic mimicry and has been described for numerous enveloped viruses (
      • Moller-Tank S.
      • Maury W.
      Phosphatidylserine receptors: Enhancers of enveloped virus entry and infection.
      ,
      • Morizono K.
      • Chen I.S.
      Role of phosphatidylserine receptors in enveloped virus infection.
      ). The PS receptor Axl, a member of the TAM family of PS receptors, was recently demonstrated to enhance the entry of infectious SARS-CoV-2 and SARS-CoV-2 pseudotyped lentivirus (
      • Wang S.
      • Qiu Z.
      • Hou Y.
      • Deng X.
      • Xu W.
      • Zheng T.
      • Wu P.
      • Xie S.
      • Bian W.
      • Zhang C.
      AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells.
      ). Such Axl-dependent entry was also shown to occur in ACE2 knockout H1299 cells, potentially indicating an ACE2-independent entry route that relies on Axl (
      • Wang S.
      • Qiu Z.
      • Hou Y.
      • Deng X.
      • Xu W.
      • Zheng T.
      • Wu P.
      • Xie S.
      • Bian W.
      • Zhang C.
      AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells.
      ). Another relevant PS receptor is the T-cell immunoglobulin and mucin domain type-1 (TIM-1) protein, which has been shown to enhance the entry of several enveloped viruses (
      • Evans J.P.
      • Liu S.-L.
      Multifaceted roles of TIM-family proteins in virus–host interactions.
      ,
      • Moller-Tank S.
      • Kondratowicz A.S.
      • Davey R.A.
      • Rennert P.D.
      • Maury W.
      Role of the phosphatidylserine receptor TIM-1 in enveloped-virus entry.
      ,
      • Moller-Tank S.
      • Albritton L.M.
      • Rennert P.D.
      • Maury W.
      Characterizing functional domains for TIM-mediated enveloped virus entry.
      ), and is a major cofactor facilitating ebolavirus endosomal entry (
      • Brunton B.
      • Rogers K.
      • Phillips E.K.
      • Brouillette R.B.
      • Bouls R.
      • Butler N.S.
      • Maury W.
      TIM-1 serves as a receptor for Ebola virus in vivo, enhancing viremia and pathogenesis.
      ,
      • Dragovich M.A.
      • Fortoul N.
      • Jagota A.
      • Zhang W.
      • Schutt K.
      • Xu Y.
      • Sanabria M.
      • Moyer D.M.
      • Moller-Tank S.
      • Maury W.
      Biomechanical characterization of TIM protein–mediated Ebola virus–host cell adhesion.
      ). Of note, TIM-1 failed to enhance the entry of SARS-CoV pseudotyped virus, but this was only tested in refractory cell lines (
      • Jemielity S.
      • Wang J.J.
      • Chan Y.K.
      • Ahmed A.A.
      • Li W.
      • Monahan S.
      • Bu X.
      • Farzan M.
      • Freeman G.J.
      • Umetsu D.T.
      TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine.
      ). As TIM-1 is not expected to act as a bona fide receptor (
      • Evans J.P.
      • Liu S.-L.
      Multifaceted roles of TIM-family proteins in virus–host interactions.
      ), it is unsurprising that TIM-1 did not render refractory cell lines permissible to SARS-CoV pseudotyped virus entry, and TIM-1 may still have an effect on SARS-CoV entry in higher ACE2-expressing cell lines. A recent report has found that TIM-1 does enhance the uptake of SARS-CoV-2 S incorporating lipid nanoparticles and this enhancement could be blocked by TIM-1 antibody blockade (
      • Ichimura T.
      • Mori Y.
      • Aschauer P.
      • Das K.M.P.
      • Padera R.F.
      • Weins A.
      • Nasr M.L.
      • Bonventre J.V.
      KIM-1/TIM-1 is a receptor for SARS-CoV-2 in lung and kidney.
      ). However, these nanoparticles lack PS, raising the question of what might be mediating this uptake. Microscale thermophoresis data suggested that TIM-1 directly interacts with SARS-CoV-2 S (
      • Ichimura T.
      • Mori Y.
      • Aschauer P.
      • Das K.M.P.
      • Padera R.F.
      • Weins A.
      • Nasr M.L.
      • Bonventre J.V.
      KIM-1/TIM-1 is a receptor for SARS-CoV-2 in lung and kidney.
      ), providing one possible explanation. Further investigation is needed to determine the role of TIM-1 on SARS-CoV-2 entry, the importance of the PS-binding activity of TIM-1, as well as the effect of TIM-1 on different SARS-CoV-2 entry routes. Given that TIM-1 has been shown to block the release of HIV-1 by trapping PS-incorporating virions on the virus producer cell (
      • Evans J.P.
      • Liu S.-L.
      Multifaceted roles of TIM-family proteins in virus–host interactions.
      ,
      • Li M.
      • Ablan S.D.
      • Miao C.
      • Zheng Y.-M.
      • Fuller M.S.
      • Rennert P.D.
      • Maury W.
      • Johnson M.C.
      • Freed E.O.
      • Liu S.-L.
      TIM-family proteins inhibit HIV-1 release.
      ,
      • Li M.
      • Waheed A.A.
      • Yu J.
      • Zeng C.
      • Chen H.-Y.
      • Zheng Y.-M.
      • Feizpour A.
      • Reinhard B.M.
      • Gummuluru S.
      • Lin S.
      TIM-mediated inhibition of HIV-1 release is antagonized by Nef but potentiated by SERINC proteins.
      ), it would be interesting to investigate the role of TIM-1, if any, on SARS-CoV-2 release.
      Another important factor promoting viral entry is heparan sulfate proteoglycans (HSPGs). Similar to PS receptors, this class of extracellular matrix glycosaminoglycans can enhance the entry of many viruses (
      • Cagno V.
      • Tseligka E.D.
      • Jones S.T.
      • Tapparel C.
      Heparan sulfate proteoglycans and viral attachment: True receptors or adaptation bias?.
      ). Previous studies have demonstrated that the introduction of cell-free HSPG reduced HCoV-NL63 attachment and replication (
      • Milewska A.
      • Zarebski M.
      • Nowak P.
      • Stozek K.
      • Potempa J.
      • Pyrc K.
      Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells.
      ). In addition, treatment of MHV, a model coronavirus, with heparin, which is closely related to HSPG (
      • Shriver Z.
      • Capila I.
      • Venkataraman G.
      • Sasisekharan R.
      Heparin and heparan sulfate: Analyzing structure and microheterogeneity.
      ), was shown to reduce virus uptake and replication, similar to treatment of target cells with heprinase (
      • De Haan C.A.
      • Li Z.
      • Te Lintelo E.
      • Bosch B.J.
      • Haijema B.J.
      • Rottier P.J.
      Murine coronavirus with an extended host range uses heparan sulfate as an entry receptor.
      ). Treatment with heprinase or exogenous heparin also reduced SARS-CoV pseudotyped virus infection and blocked binding of soluble SARS-CoV S to target cells (
      • Lang J.
      • Yang N.
      • Deng J.
      • Liu K.
      • Yang P.
      • Zhang G.
      • Jiang C.
      Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans.
      ). All these results suggest that the mechanism of HSPG-mediated enhancement of coronavirus infection may be the result of direct interaction between HSPG and Spike. For SARS-CoV-2, recent reports based on surface plasmon resonance, circular dicromism, ELISA, sepharose pulldown, and microarray data have established that SARS-CoV-2 S interacts with HSPG and heparin and that binding heparin may induce a conformational change in S (
      • Mycroft-West C.J.
      • Su D.
      • Elli S.
      • Li Y.
      • Guimond S.E.
      • Miller G.J.
      • Turnbull J.E.
      • Yates E.A.
      • Guerrini M.
      • Fernig D.G.
      The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 receptor binding domain undergoes conformational change upon heparin binding.
      ,
      • Liu L.
      • Chopra P.
      • Li X.
      • Wolfert M.A.
      • Tompkins S.M.
      • Boons G.-J.
      SARS-CoV-2 spike protein binds heparan sulfate in a length-and sequence-dependent manner.
      ,
      • Clausen T.M.
      • Sandoval D.R.
      • Spliid C.B.
      • Pihl J.
      • Perrett H.R.
      • Painter C.D.
      • Narayanan A.
      • Majowicz S.A.
      • Kwong E.M.
      • McVicar R.N.
      SARS-CoV-2 infection depends on cellular heparan sulfate and ACE2.
      ,
      • Zhang Q.
      • Chen C.Z.
      • Swaroop M.
      • Xu M.
      • Wang L.
      • Lee J.
      • Wang A.Q.
      • Pradhan M.
      • Hagen N.
      • Chen L.
      Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro.
      ,
      • Kim S.Y.
      • Jin W.
      • Sood A.
      • Montgomery D.W.
      • Grant O.C.
      • Fuster M.M.
      • Fu L.
      • Dordick J.S.
      • Woods R.J.
      • Zhang F.
      Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions.
      ). Heparin has also been shown to block the entry of SARS-CoV-2 pseudotyped vesicular stomatitis virus (
      • Clausen T.M.
      • Sandoval D.R.
      • Spliid C.B.
      • Pihl J.
      • Perrett H.R.
      • Painter C.D.
      • Narayanan A.
      • Majowicz S.A.
      • Kwong E.M.
      • McVicar R.N.
      SARS-CoV-2 infection depends on cellular heparan sulfate and ACE2.
      ) and murine leukemia virus (
      • Zhang Q.
      • Chen C.Z.
      • Swaroop M.
      • Xu M.
      • Wang L.
      • Lee J.
      • Wang A.Q.
      • Pradhan M.
      • Hagen N.
      • Chen L.
      Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro.
      ) as well as infectious SARS-CoV-2 (
      • Clausen T.M.
      • Sandoval D.R.
      • Spliid C.B.
      • Pihl J.
      • Perrett H.R.
      • Painter C.D.
      • Narayanan A.
      • Majowicz S.A.
      • Kwong E.M.
      • McVicar R.N.
      SARS-CoV-2 infection depends on cellular heparan sulfate and ACE2.
      ,
      • Kwon P.S.
      • Oh H.
      • Kwon S.-J.
      • Jin W.
      • Zhang F.
      • Fraser K.
      • Hong J.J.
      • Linhardt R.J.
      • Dordick J.S.
      Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro.
      ). Other sulfated polysaccharides have also been shown to disrupt infection by SARS-CoV-2 pseudotyped virus and infectious SARS-CoV-2 (
      • Kwon P.S.
      • Oh H.
      • Kwon S.-J.
      • Jin W.
      • Zhang F.
      • Fraser K.
      • Hong J.J.
      • Linhardt R.J.
      • Dordick J.S.
      Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro.
      ,
      • Tandon R.
      • Sharp J.S.
      • Zhang F.
      • Pomin V.H.
      • Ashpole N.M.
      • Mitra D.
      • McCandless M.G.
      • Jin W.
      • Liu H.
      • Sharma P.
      Effective inhibition of SARS-CoV-2 entry by heparin and enoxaparin derivatives.
      ). Furthermore, the development of inhibitors to block the interaction of SARS-CoV-2 S with HSPGs has been proposed as a potential therapeutic strategy for combating COVID-19 (
      • Zhang Q.
      • Chen C.Z.
      • Swaroop M.
      • Xu M.
      • Wang L.
      • Lee J.
      • Wang A.Q.
      • Pradhan M.
      • Hagen N.
      • Chen L.
      Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro.
      ,
      • Kwon P.S.
      • Oh H.
      • Kwon S.-J.
      • Jin W.
      • Zhang F.
      • Fraser K.
      • Hong J.J.
      • Linhardt R.J.
      • Dordick J.S.
      Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro.
      ). Collectively, this evidence suggests that HSPG may serve as an entry cofactor for SARS-CoV-2; however, the mechanism of action as well as its role in virus spread and pathogenesis remains unclear and warrants further investigation.
      When not serving as the primary coronavirus receptor as for HCoV-HKU1, HCoV-OC43, and Bovine-CoV (
      • Huang X.
      • Dong W.
      • Milewska A.
      • Golda A.
      • Qi Y.
      • Zhu Q.K.
      • Marasco W.A.
      • Baric R.S.
      • Sims A.C.
      • Pyrc K.
      Human coronavirus HKU1 spike protein uses O-acetylated sialic acid as an attachment receptor determinant and employs hemagglutinin-esterase protein as a receptor-destroying enzyme.
      ,
      • Schultze B.
      • Gross H.
      • Brossmer R.
      • Herrler G.
      The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant.
      ,
      • Hulswit R.J.
      • Lang Y.
      • Bakkers M.J.
      • Li W.
      • Li Z.
      • Schouten A.
      • Ophorst B.
      • Van Kuppeveld F.J.
      • Boons G.-J.
      • Bosch B.-J.
      Human coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved receptor-binding site in spike protein domain A.
      ), sialic acids can still act as important coronavirus entry cofactors. These abundant cell-surface glycans serve as receptors and attachment cofactors for a wide range of viruses, including influenza (
      • Neu U.
      • Bauer J.
      • Stehle T.
      Viruses and sialic acids: Rules of engagement.
      ,
      • Schwegmann-Weßels C.
      • Herrler G.
      Sialic acids as receptor determinants for coronaviruses.
      ). The S protein of MERS-CoV has been shown to hemagglutinate red blood cells (
      • Li W.
      • Hulswit R.J.
      • Widjaja I.
      • Raj V.S.
      • McBride R.
      • Peng W.
      • Widagdo W.
      • Tortorici M.A.
      • Van Dieren B.
      • Lang Y.
      Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.
      ), which are highly decorated with sialic acid moieties, and MERS-CoV S-coated nanoparticles can bind to sialic acid moieties (
      • Li W.
      • Hulswit R.J.
      • Widjaja I.
      • Raj V.S.
      • McBride R.
      • Peng W.
      • Widagdo W.
      • Tortorici M.A.
      • Van Dieren B.
      • Lang Y.
      Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.
      ). In addition, the structure of MERS-CoV S-interacting with the sialic acid Neu5Ac has been solved (
      • Park Y.-J.
      • Walls A.C.
      • Wang Z.
      • Sauer M.M.
      • Li W.
      • Tortorici M.A.
      • Bosch B.-J.
      • DiMaio F.
      • Veesler D.
      Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors.
      ). Of importance, neuraminidase digestion of sialic acid moieties on Calu-3 cells reduces MERS-CoV replication (
      • Li W.
      • Hulswit R.J.
      • Widjaja I.
      • Raj V.S.
      • McBride R.
      • Peng W.
      • Widagdo W.
      • Tortorici M.A.
      • Van Dieren B.
      • Lang Y.
      Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.
      ). However, it has recently been reported that SARS-CoV-2 S failed to bind any residues in a sialic acid microarray (
      • Hao W.
      • Ma B.
      • Li Z.
      • Wang X.
      • Gao X.
      • Li Y.
      • Qin B.
      • Shang S.
      • Cui S.
      • Tan Z.
      Binding of the SARS-CoV-2 spike protein to glycans.
      ), indicating that a more sensitive assay may be required to detect SARS-CoV-2 S binding of sialic acids.
      The C-type lectins, L-SIGN and DC-SIGN, also play an important role in enhancing viral entry (
      • Alvarez C.P.
      • Lasala F.
      • Carrillo J.
      • Muñiz O.
      • Corbí A.L.
      • Delgado R.
      C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans.
      ,
      • Cambi A.
      • de Lange F.
      • van Maarseveen N.M.
      • Nijhuis M.
      • Joosten B.
      • van Dijk E.M.
      • de Bakker B.R.I.
      • Fransen J.A.
      • Bovee-Geurts P.H.
      • van Leeuwen F.N.
      Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells.
      ,
      • Lozach P.-Y.
      • Amara A.
      • Bartosch B.
      • Virelizier J.-L.
      • Arenzana-Seisdedos F.
      • Cosset F.-L.
      • Altmeyer R.
      C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles.
      ). The S protein of SARS-CoV has been shown to bind DC-SIGN (
      • Marzi A.
      • Gramberg T.
      • Simmons G.
      • Möller P.
      • Rennekamp A.J.
      • Krumbiegel M.
      • Geier M.
      • Eisemann J.
      • Turza N.
      • Saunier B.
      DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus.
      ), and DC-SIGN can enhance the entry of SARS-CoV S pseudotyped lentivirus particles (
      • Yang Z.-Y.
      • Huang Y.
      • Ganesh L.
      • Leung K.
      • Kong W.-P.
      • Schwartz O.
      • Subbarao K.
      • Nabel G.J.
      pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN.
      ,
      • Thepaut M.
      • Luczkowiak J.
      • Vives C.
      • Labiod N.
      • Bally I.
      • Lasala F.
      • Grimoire Y.
      • Fenel D.
      • Sattin S.
      • Thielens N.
      • Schoehn G.
      • Bernardi A.
      • Delgado R.
      • Fieschi F.
      DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist.
      ) with specific S glycosylation site mutants failing to facilitate SARS-CoV S pseudotyped virus entry into HeLa cells overexpressing L-SIGN or DC-SIGN (
      • Han D.P.
      • Lohani M.
      • Cho M.W.
      Specific asparagine-linked glycosylation sites are critical for DC-SIGN-and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry.
      ). Similarly, it has been recently reported that SARS-CoV-2 S can bind both DC-SIGN and L-SIGN and exogenous expression of either DC-SIGN or L-SIGN can enhance SARS-CoV-2 pseudotyped virus entry into HEK 293T cells (
      • Amraei R.
      • Napoleon M.
      • Yin W.
      • Berrigan J.
      • Suder E.
      • Zhao G.
      • Olejnik J.
      • Gummuluru S.
      • Muhlberger E.
      • Chitalia V.
      CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2 and are differentially expressed in lung and kidney epithelial and endothelial cells.
      ). Although these cellular factors are claimed as bone fide receptors for SARS-CoV-2 (
      • Amraei R.
      • Napoleon M.
      • Yin W.
      • Berrigan J.
      • Suder E.
      • Zhao G.
      • Olejnik J.
      • Gummuluru S.
      • Muhlberger E.
      • Chitalia V.
      CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2 and are differentially expressed in lung and kidney epithelial and endothelial cells.
      ,
      • Brufsky A.
      • Lotze M.T.
      DC/L-SIGNs of hope in the COVID-19 pandemic.
      ), available data seem to suggest a role as attachment cofactors. Thus, probing the effect of L-SIGN/DC-SIGN on SARS-CoV-2 entry of ACE2 knockout cells should help clarify their role.
      A better understanding of the cellular cofactors enhancing SARS-CoV-2 entry, as discussed above, will further elucidate SARS-CoV-2 tissue and host tropism. In addition, such an understanding may allow for the development of therapeutics disrupting the entry phase of the SARS-CoV-2 lifecycle. However, all these entry cofactors require further investigation to clarify the mechanisms for enhancement of SARS-CoV-2 entry and to distinguish entry cofactors from bona fide receptors, especially in physiologically relevant systems.

      Entry restriction factors: Another side of the coin

      Host restriction factors are another set of important modulators that can act on different steps of viral replication (
      • Kluge S.F.
      • Sauter D.
      • Kirchhoff F.
      SnapShot: Antiviral restriction factors.
      ,
      • Duggal N.K.
      • Emerman M.
      Evolutionary conflicts between viruses and restriction factors shape immunity.
      ). Although they are typically induced by type I interferon and often antagonized by a viral factor to allow evasion of host restriction, some exceptions do exist, such as serine incorporator (SERINC) proteins, which impair HIV infectivity (
      • Kluge S.F.
      • Sauter D.
      • Kirchhoff F.
      SnapShot: Antiviral restriction factors.
      ,
      • Duggal N.K.
      • Emerman M.
      Evolutionary conflicts between viruses and restriction factors shape immunity.
      ). Some of these restriction factors can inhibit viral entry and could also have additional or even opposite functions on other steps of viral replication, such as assembly and release. Here we focus on recent investigations into the roles of interferon-induced transmembrane (IFITM) proteins and lymphocyte antigen 6 complex locus E (Ly6E) in restriction of SARS-CoV-2 entry (Table 1).
      The IFITM proteins are a class of interferon-stimulated genes (ISGs) that block viral membrane fusion with target cells (
      • Li K.
      • Markosyan R.M.
      • Zheng Y.-M.
      • Golfetto O.
      • Bungart B.
      • Li M.
      • Ding S.
      • He Y.
      • Liang C.
      • Lee J.C.
      IFITM proteins restrict viral membrane hemifusion.
      ,
      • Zhao X.
      • Li J.
      • Winkler C.A.
      • An P.
      • Guo J.-T.
      IFITM genes, variants, and their roles in the control and pathogenesis of viral infections.
      ). These restriction factors have a complicated role in coronavirus replication. It has been demonstrated that knockdown of IFITM3 enhanced entry of HCoV-NL63, HCoV-229E, SARS-CoV, and MERS-CoV S bearing pseudotyped virus (
      • Wrensch F.
      • Winkler M.
      • Pöhlmann S.
      IFITM proteins inhibit entry driven by the MERS-coronavirus spike protein: Evidence for cholesterol-independent mechanisms.
      ,
      • Huang I.-C.
      • Bailey C.C.
      • Weyer J.L.
      • Radoshitzky S.R.
      • Becker M.M.
      • Chiang J.J.
      • Brass A.L.
      • Ahmed A.A.
      • Chi X.
      • Dong L.
      Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.
      ). In addition, overexpression of IFITM1 to 3 inhibited infectious SARS-CoV replication and pseudotyped virus entry (
      • Huang I.-C.
      • Bailey C.C.
      • Weyer J.L.
      • Radoshitzky S.R.
      • Becker M.M.
      • Chiang J.J.
      • Brass A.L.
      • Ahmed A.A.
      • Chi X.
      • Dong L.
      Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.
      ). However, IFITM3 has been shown to enhance the entry of HCoV-OC43 (
      • Zhao X.
      • Guo F.
      • Liu F.
      • Cuconati A.
      • Chang J.
      • Block T.M.
      • Guo J.-T.
      Interferon induction of IFITM proteins promotes infection by human coronavirus OC43.
      ), and disruption of the N-terminal domain of IFITM3, specifically at tyrosine 20 (Y20) in an endosomal sorting motif, has been shown to allow IFITM3 to enhance the entry of SARS-CoV and MERS-CoV (
      • Zhao X.
      • Sehgal M.
      • Hou Z.
      • Cheng J.
      • Shu S.
      • Wu S.
      • Guo F.
      • Le Marchand S.J.
      • Lin H.
      • Chang J.
      Identification of residues controlling restriction versus enhancing activities of IFITM proteins on entry of human coronaviruses.
      ). These dual roles of IFITMs in coronavirus entry are reflected in recent SARS-CoV-2 studies. For instance, knockdown of IFITMs in Calu-3 or Caco-2 cells enhanced entry, whereas overexpression of IFITMs inhibited entry in HEK 293T cells overexpressing ACE2 (HEK 293T-ACE2) (
      • Bozzo C.P.
      • Nchioua R.
      • Volcic M.
      • Wettstein L.
      • Weil T.
      • Krüger J.
      • Heller S.
      • Conzelmann C.
      • Mueller J.A.
      • Gross R.
      IFITM proteins promote SARS-CoV-2 infection in human lung cells.
      ,
      • Shi G.
      • Kenney A.D.
      • Kudryashova E.
      • Zani A.
      • Zhang L.
      • Lai K.K.
      • Hall-Stoodley L.
      • Robinson R.T.
      • Kudryashov D.S.
      • Compton A.A.
      Opposing activities of IFITM proteins in SARS-CoV-2 infection.
      ). Similarly, overexpression of IFITMs inhibited infectious SARS-CoV-2 replication in HEK 293T-ACE2 (
      • Bozzo C.P.
      • Nchioua R.
      • Volcic M.
      • Wettstein L.
      • Weil T.
      • Krüger J.
      • Heller S.
      • Conzelmann C.
      • Mueller J.A.
      • Gross R.
      IFITM proteins promote SARS-CoV-2 infection in human lung cells.
      ,
      • Shi G.
      • Kenney A.D.
      • Kudryashova E.
      • Zani A.
      • Zhang L.
      • Lai K.K.
      • Hall-Stoodley L.
      • Robinson R.T.
      • Kudryashov D.S.
      • Compton A.A.
      Opposing activities of IFITM proteins in SARS-CoV-2 infection.
      ). Overexpression of IFITMs, especially IFITM1, inhibits syncytia formation of cells overexpressing ACE2 with cells expressing SARS-CoV-2 S, and expression of TMPRSS2 can counteract this inhibitory effect (
      • Buchrieser J.
      • Dufloo J.
      • Hubert M.
      • Monel B.
      • Planas D.
      • Rajah M.M.
      • Planchais C.
      • Porrot F.
      • Guivel-Benhassine F.
      • Van der Werf S.
      Syncytia formation by SARS-CoV-2-infected cells.
      ). However, knockdown of IFITMs, especially IFITM2, in the more biologically relevant Calu-3 cell line decreased infectious SARS-CoV-2 replication (
      • Bozzo C.P.
      • Nchioua R.
      • Volcic M.
      • Wettstein L.
      • Weil T.
      • Krüger J.
      • Heller S.
      • Conzelmann C.
      • Mueller J.A.
      • Gross R.
      IFITM proteins promote SARS-CoV-2 infection in human lung cells.
      ). In addition, introduction of a Y20A mutation in IFITM3 converted IFITM3 into an enhancer of infectious SARS-CoV-2 replication in HEK 293T-ACE2 cells (
      • Shi G.
      • Kenney A.D.
      • Kudryashova E.
      • Zani A.
      • Zhang L.
      • Lai K.K.
      • Hall-Stoodley L.
      • Robinson R.T.
      • Kudryashov D.S.
      • Compton A.A.
      Opposing activities of IFITM proteins in SARS-CoV-2 infection.
      ). It is also interesting to note that overexpression of TMPRSS2 allowed for IFITM3 restriction of SARS-CoV-2 entry to be overcome (
      • Shi G.
      • Kenney A.D.
      • Kudryashova E.
      • Zani A.
      • Zhang L.
      • Lai K.K.
      • Hall-Stoodley L.
      • Robinson R.T.
      • Kudryashov D.S.
      • Compton A.A.
      Opposing activities of IFITM proteins in SARS-CoV-2 infection.
      ). Our unpublished data showed that the effect of IFITMs on viral entry is cell type dependent and may be related to the level and stability of ACE2 expression on the plasma membrane (Qu et al., unpublished data). Given that mutation of an endosomal sorting motif in IFITM3 ablates its restriction of SARS-CoV-2 and that expression of TMPRSS2, presumed to enhance SARS-CoV-2 entry at the cell membrane, can overcome IFITM3 restriction, these results indicate that IFITM3 likely restricts SARS-CoV-2 entry primarily in the endosome, which could be modulated by other cellular cofactors. The mechanism for IFITM-mediated differential effects on SARS-CoV-2 entry and replication in physiologically relevant cells requires further investigation.
      Ly6/uPAR family member lymphocyte antigen 6 complex locus E (Ly6E) is an interferon-inducible ISG that has previously been shown to enhance viral entry for several enveloped viruses, including HIV (
      • Mar K.B.
      • Rinkenberger N.R.
      • Boys I.N.
      • Eitson J.L.
      • McDougal M.B.
      • Richardson R.B.
      • Schoggins J.W.
      LY6E mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step.
      ,
      • Yu J.
      • Liang C.
      • Liu S.-L.
      Interferon-inducible LY6E protein promotes HIV-1 infection.
      ). However, for HCoV-OC43, overexpression of Ly6E was shown to inhibit virus entry and infection in HEK293 and A549 cells, whereas knockdown of Ly6E enhanced viral infection in HepG2 cells (
      • Zhao X.
      • Zheng S.
      • Chen D.
      • Zheng M.
      • Li X.
      • Li G.
      • Lin H.
      • Chang J.
      • Zeng H.
      • Guo J.-T.
      LY6E restricts entry of human coronaviruses, including currently pandemic SARS-CoV-2.
      ). In addition, overexpression of Ly6E was shown to inhibit entry of lentiviral pseudotyped virus bearing S from HCoV-OC43, HCoV-229E, HCoV-NL63, MERS-CoV, or SARS-CoV-2 (
      • Zhao X.
      • Zheng S.
      • Chen D.
      • Zheng M.
      • Li X.
      • Li G.
      • Lin H.
      • Chang J.
      • Zeng H.
      • Guo J.-T.
      LY6E restricts entry of human coronaviruses, including currently pandemic SARS-CoV-2.
      ,
      • Pfaender S.
      • Mar K.B.
      • Michailidis E.
      • Kratzel A.
      • Boys I.N.
      • V’kovski P.
      • Fan W.
      • Kelly J.N.
      • Hirt D.
      • Ebert N.
      • Stalder H.
      • Kleine-Weber H.
      • Hoffmann M.
      • Hoffmann H.-H.
      • Saeed M.
      • et al.
      LY6E impairs coronavirus fusion and confers immune control of viral disease.
      ) and inhibited infection of their replication-competent viruses (
      • Pfaender S.
      • Mar K.B.
      • Michailidis E.
      • Kratzel A.
      • Boys I.N.
      • V’kovski P.
      • Fan W.
      • Kelly J.N.
      • Hirt D.
      • Ebert N.
      • Stalder H.
      • Kleine-Weber H.
      • Hoffmann M.
      • Hoffmann H.-H.
      • Saeed M.
      • et al.
      LY6E impairs coronavirus fusion and confers immune control of viral disease.
      ). Finally, Ly6E knockout mice exhibited more severe MHV infection (
      • Pfaender S.
      • Mar K.B.
      • Michailidis E.
      • Kratzel A.
      • Boys I.N.
      • V’kovski P.
      • Fan W.
      • Kelly J.N.
      • Hirt D.
      • Ebert N.
      • Stalder H.
      • Kleine-Weber H.
      • Hoffmann M.
      • Hoffmann H.-H.
      • Saeed M.
      • et al.
      LY6E impairs coronavirus fusion and confers immune control of viral disease.
      ). Although these results indicate Ly6E is a broadly acting restriction factor for coronaviruses that acts on virus entry, the effect can be cell type dependent, as we have shown for HIV (
      • Yu J.
      • Liang C.
      • Liu S.-L.
      Interferon-inducible LY6E protein promotes HIV-1 infection.
      ,
      • Yu J.
      • Liang C.
      • Liu S.-L.
      CD4-dependent modulation of HIV-1 entry by LY6E.
      ).
      Owing to space limitation, we only highlight here the IFITM and Ly6E family proteins, with published effects on SARS-CoV-2, to demonstrate the role of ISGs in limiting coronavirus infection and pathogenesis; there are additional host restriction factors, including TRIM56 and tetherin (
      • Liu B.
      • Li N.L.
      • Wang J.
      • Shi P.-Y.
      • Wang T.
      • Miller M.A.
      • Li K.
      Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against flaviviruses and coronavirus.
      ,
      • Wang S.M.
      • Huang K.J.
      • Wang C.T.
      Severe acute respiratory syndrome coronavirus spike protein counteracts BST2-mediated restriction of virus-like particle release.
      ), that critically regulate different steps of replication for other coronaviruses. Of importance, viral proteins or antagonists have been reported to counteract some of these host restriction factors, including for SARS-CoV (
      • Wang S.M.
      • Huang K.J.
      • Wang C.T.
      Severe acute respiratory syndrome coronavirus spike protein counteracts BST2-mediated restriction of virus-like particle release.
      ). It is this mode of virus–host evolutionary arms race that drives their coadaptation, which accounts in part for the emergence of new variants or species that spills over from animals to humans resulting in endemic or pandemic of infectious diseases such as COVID-19 (
      • Duggal N.K.
      • Emerman M.
      Evolutionary conflicts between viruses and restriction factors shape immunity.
      ,
      • Daugherty M.D.
      • Malik H.S.
      Rules of engagement: Molecular insights from host-virus arms races.
      ,
      • Wong L.-Y.R.
      • Lui P.-Y.
      • Jin D.-Y.
      A molecular arms race between host innate antiviral response and emerging human coronaviruses.
      ).

      Concluding remarks

      Coronavirus entry is characterized by a plasticity of entry routes regulated by receptor utilization, cofactor modulation, protease cleavage, as well as host restriction factors. Although much progress has been made in each of these aspects, there is a need for more investigation of their mechanisms of action, especially with respect to SARS-CoV-2 entry and how these factors interact with each other and ultimately dictate the entry pathways, i.e., endosomal or plasma membrane routes. Understanding the complex mechanisms of SARS-CoV-2 entry may inform strategies for the design of effective therapeutics to disrupt the virus life cycle. More broadly and also significantly, a better grasp of coronavirus entry will help inform our understanding of the zoonotic transmission, spillover, and pandemic potential of future emerging coronaviruses.

      Conflict of interest

      The authors declare no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank members of the Liu lab at The Ohio State University for helpful discussions.

      Author contributions

      J. P. E. and S.-L. L. conceptualization; J. P. E. writing-original draft; J. P. E. and S.-L. L. writing-review and editing.

      Funding and additional information

      J. P. E. was supported by Glenn Barber Fellowship . S.-L. L. was supported in part by a fund provided by an anonymous private donor to OSU, as well as by NIH grant R01 AI150473 and by the National Cancer Institute of the NIH under award no. U54CA260582. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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