A protein produced by the human immune system can strongly inhibit corona viruses, including SARS-Cov-2, the pathogen causing COVID-19.
An international team from Germany, Switzerland and the U.S. successfully showed that the LY6E-Protein prevents coronaviruses from causing an infection.
“This finding might lead to the development of new therapeutic approaches against coronaviruses,” says Professor Stephanie Pfänder from the Department for Molecular and Medical Virology at Ruhr-Universität Bochum (RUB), lead author of the study published by the team in the journal Nature Microbiology on 23 July 2020.
Strengthening influenza viruses, impairing corona viruses
The LY6E protein plays a role in various diseases: US researchers Professor John Schoggins and Professor Charles Rice discovered that the protein enhances the infectivity of influenza viruses. In contrast, coronaviruses are inhibited by LY6E.
Funded by a Marie Curie Individual Fellowship of the European Union, Stephanie Pfänder, who was then working at the Institute of Virology and Immunology in Switzerland, visited Charles Rice’s lab at Rockefeller University in New York in 2017, in order to identify genes that prevent coronavirus infections.
“This led to the discovery that LY6E has the opposite effect on coronaviruses compared to influenza viruses,” explains the researcher.
Further investigations showed that the protein exerted this inhibitory effect on all analyzed coronaviruses, including the pathogens causing SARS and Mers as well as SARS-Cov-2 which causes COVID-19.
Viruses unable to fuse
Tests with different cell cultures showed that LY6E affects the ability of the virus to fuse with the host cells.
“If the virus is unable to fuse with these cells, it can’t cause infection,” explains corresponding author Professor Volker Thiel from the University of Bern.
The validation in an animal model succeeded thanks to a collaboration with the laboratory of John Schoggins at the Southwestern Medical Center of the University of Texas.
The experiments conducted there led to the discovery that the mouse variant of the protein called Ly6e is crucial for the protection of immune cells against infections.
In the absence of Ly6e, immune cells such as dendritic cells and B-cells become more susceptible to infection and their numbers decrease dramatically. Mice lacking Ly6e in immune cells are highly susceptible to a normally non-lethal mouse coronavirus and succumb to infection.
Understanding basic concepts
The researchers point out that the mouse coronavirus used in the experiment differs significantly from the pathogen causing the current COVID-19 outbreak – for example, it causes hepatisis rather than respiratory disease.
Nevertheless, it is widely accepted as a model for understanding the basic concepts of coronavirus replication and immune responses in a living animal.
“Our study provides new insights into how important these antiviral genes are for the control of viral infection and for an adequate immune response against the virus,” say the authors.
“Since LY6E is a naturally occurring human protein, we hope that this knowledge will aid the development of therapies that may one day be used to treat coronavirus infections.” A therapeutic approach that mimics the mechanism of action of LY6E may provide a first line of defense against novel coronavirus infections.
Screening a cDNA library of >350 human interferon-stimulated genes for antiviral activity against endemic human coronavirus HCoV-229E (associated with the common cold), Pfaender S & Mar K et al. identify lymphocyte antigen 6 complex, locus E (Ly6E) as an inhibitor of cellular infection of Huh7 cells, a human hepatoma cell line susceptible to HCoV-229E and other coronaviruses.
In a series of consecutive in vitro experiments including both stable Ly6E overexpression and CRISPR-Cas9-mediated knockout the authors further demonstrate that Ly6E reduces cellular infection by various other coronaviruses including human SARS-CoV and SARS-CoV-2 as well as murine CoV mouse hepatitis virus (MHV).
Their experiments suggest that this effect is dependent on Ly6E inhibition of CoV strain-specific spike protein-mediated membrane fusion required for viral cell entry.
To address the function of Ly6E in vivo, hematopoietic stem cell-specific Ly6E knock-out mice were generated by breeding Ly6Efl/fl mice (referred to as functional wild-type mice) with transgenic Vav-iCre mice (offspring referred to as Ly6E HSC ko mice); wild-type and Ly6E HSC ko mice of both sexes were infected intraperitoneally with varying doses of the natural murine coronavirus MHV, a pathogen that depending on route of infection can cause a wide range of diseases in mice including hepatitis, enteritis and encephalomyelitis.
Briefly, compared to wild-type controls, mice lacking hematopoietic cell-expressed Ly6E were found to present with increased mortality, a more severe disease phenotype as based on serum ALT levels (prognostic of liver damage), liver histopathology, and viral titers in the spleen.
Moreover, bulk RNAseq analysis of infected liver and spleen tissues indicated changes in gene expression pathways related to tissue damage and antiviral immune responses as well as a reduction of genes associated with type I IFN response and inflammation.
Finally, the authors report substantial differences in the numbers of hepatic and splenic APC subsets between wild-type and knockout mice following MHV infection and show that Ly6E-deficient B cells and to a lesser extent also DCs are particularly susceptible to MHV infection in vitro.
Experiments and data in this study are presented in an overall logical and coherent fashion; however, some observations and the conclusions drawn are problematic and should be further addressed & discussed by the authors.
Methodological & formal limitations include relatively low replicate numbers as well as missing technical replicates for some in vitro experiments (cf. Fig. legend 1; Fig. legend 2e); the omission of “outliers” in Fig. legend 2 without an apparent rationale as to why this approach was chosen; the lack of detection of actual Ly6E protein levels in Ly6E HSC ko or wild-type mice; and most importantly, missing information on RNAseq data collection & analysis in the method section and throughout the paper.
A more relevant concern though is that the interpretation of the experimental data presented and the language used tend to overrate and at times overgeneralize findings: for example, while the authors demonstrate statistically significant, Ly6E-mediated reduction of coronavirus titers in stable cells lines in vitro, it remains unclear whether a viral titer reduction by one log decade would be of actual biological relevance in face of high viral titers in vivo.
After high-dose intraperitoneal MHV infection in vivo, early viral titers in Ly6E HSC knockout vs. wt mice showed an elevation in the spleen (~1.5 log decades) but not liver of the ko mice (other tissue not evaluated), and while ko mice presented with only modestly increased liver pathology, both male and female ko mice exhibited significantly higher mortality.
Thus, the manuscript title statement that “Ly6E … confers immune control of viral disease” is directly supported by only limited in vivo data, and gain-of-function experiments (eg. Ly6E overexpression) were not performed.
Of additional note here, tissue tropism and virulence differ greatly among various MHV strains and isolates whereas dose, route of infection, age, genetic background and sex of the mice used may additionally affect disease outcome and phenotype (cf. Taguchi F & Hirai-Yuki A, https://doi.org/10.3389/fmi…
Kanolkhar A et al, https://jvi.asm.org/content/ 83/18/9258). Observations attributed to hematopoietic stem cell-specific Ly6E deletion could therefore be influenced by the different genetic backgrounds of floxed and cre mice used, and although it appears that littermates wt and ko littermates were used in the experiments, the potentially decisive impact of strain differences should at least have been discussed.
Along these lines, it should also be taken into account that the majority of human coronaviruses cause respiratory symptoms, which follow a different clinical course engaging other primary cellular mediators than the hepatotropic murine MHV disease studied here.
It therefore remains highly speculative how the findings reported in this study will translate to human disease and it would therefore be important to test other routes of MHV infection and doses that have been described to produce a more comparable phenotype to human coronavirus disease (cf. Kanolkhar A et al, https://jvi.asm.org/content/ 83/18/9258).
Another important shortcoming of this study is the lack of any information on functional deficits or changes in Ly6E-deficient immune cells and how this might relate to the phenotype observed. Overall, the in vitro experiments are more convincing than the in vivo studies which appear somewhat limited.
Overall relevance for the field
Despite some shortcomings, the experiments performed in this study suggest a novel and somewhat unexpected role of Ly6E in the protection against coronaviruses across species.
These findings are of relevance and should be further explored in ongoing research on potential coronavirus therapies.
Yet an important caveat pertains to the authors’ suggestion that “therapeutic mimicking of Ly6E action” may constitute a first line of defense against novel coronaviruses since their own prior work demonstrated that Ly6E can enhance rather than curtail infection with influenza A and other viruses.
Reviewed as part of a project by students, postdoctoral fellows and faculty at the Immunology Institute of the Icahn School of Medicine at Mount Sinai
More information: Stephanie Pfaender et al. LY6E impairs coronavirus fusion and confers immune control of viral disease, Nature Microbiology (2020). DOI: 10.1038/s41564-020-0769-y