LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

Abstract

<jats:p>C3A is a sub-clone of human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT) and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins with identified function of interfering virus entry, expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFITM3 restriction of human coronavirus entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis <jats:italic>via</jats:italic> a mechanism distinct from other factors that modulate CoV entry.</jats:p> <jats:p><jats:bold>IMPORTANCE</jats:bold> Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control by host innate and adaptive immune responses. In the last decade, several interferon inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as host factors to facilitate the entry of several human pathogenic viruses, including human immunodeficiency virus, influenza A virus and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.</jats:p>

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells

“…The enhanced replication shown by SARS-CoV-2 with furin pre-cleaved spike to utilise TMPRSS2 to enter cells efficiently suggested this virus may prefer to enter cells via membranes near the cell surface or in the early endosome rather than be trafficked into late endosomes where it could be cleaved by host cathepsins. We hypothesised that the wild type virus may be avoiding host restriction factors in the endosome, such as IFITM2/3 proteins, which have previously been shown to be able to restrict SARS-CoV and SARS-CoV-2 entry (Huang et al, 2011; Zhao et al, 2020; Zheng et al, 2020). The antifungal agent amphotericin B (amphoB) has been well described as inhibiting the restriction imposed by IFITM proteins, particularly the endosomal/lysosomal localised IFITM2/3, potentially modulating the host membrane fluidity required for efficient restriction (Lin et al, 2013; Zhao et al, 2020; Zheng et al, 2020).…”

“…We hypothesised that the wild type virus may be avoiding host restriction factors in the endosome, such as IFITM2/3 proteins, which have previously been shown to be able to restrict SARS-CoV and SARS-CoV-2 entry (Huang et al, 2011; Zhao et al, 2020; Zheng et al, 2020). The antifungal agent amphotericin B (amphoB) has been well described as inhibiting the restriction imposed by IFITM proteins, particularly the endosomal/lysosomal localised IFITM2/3, potentially modulating the host membrane fluidity required for efficient restriction (Lin et al, 2013; Zhao et al, 2020; Zheng et al, 2020). We could also show that 293T-ACE2, Caco-2, Calu-3 and HAEs all constitutively expressed IFITM3, even in the absence of exogenous interferon (Figure 3E-H).…”

Syncytia formation by SARS-CoV-2 infected cells

“…IFITMs inhibit SARS-CoV, 229E and MERS-CoV entry, but promote infection by HCoV-OC43, a coronavirus that causes the common cold [33][34][35][36][37][38] . IFITMs, as well as other ISGs, including LY6E and Cholesterol 25-hydrolase (CH25H), impair SARS-CoV-2 replication by blocking the fusion of virions [39][40][41] . Most of the experiments regarding these ISGs have been performed with single-cycle viral pseudotypes.…”

ETV7 limits antiviral gene expression and control of SARS-CoV-2 and influenza viruses

“…4H). This enhanced control was likely mediated via the ISG LY6E, which potently restricts SARS-CoV-2 (67,68) and we found to be regulated by ETV7 (Fig. S4B).…”

“…Additionally, many of the ETV7-regulated ISGs restrict respiratory viruses (42,67,68), and we showed that loss of ETV7 can further enhance the ability of type I IFN to control influenza virus and SARS-CoV-2 replication. Further work is required to understand the complexity of IFN regulation, while therapeutic targeting of factors like ETV7 could lead to the development of a new class of host-directed antivirals that can enhance or tailor ISG responses to specific viruses.…”