Periodic Reporting for period 4 - VIREX (Mumps VIRus EXploitation of the human adhesion receptor GPR125)
Período documentado: 2021-01-01 hasta 2022-06-30
MuV causes the highly contagious mumps disease in children characterized by swollen salivary glands. In adults, it also infects gonadal tissue giving rise to orchitis and oophoritis, and in the long run causes infertility. MuV is highly neurotropic and the cause of meningitis and encephalitis, with signs of subclinical infections in up to 50% after vaccination. Like other paramyxovirinae, MuV is a small virus. It only encodes seven genes giving rise to nine proteins, one of them is the short hydrophobic (SH) protein which is a membrane protein of 57 residues (6.8 KDa). Like the SH proteins of related paramyxoviruses, MuV-SH is dispensable for virus growth in tissue culture. Nevertheless, it acts as a viral antagonist to the host's innate immune system, through inhibition tumor necrosis factor α (TNF-α) release from infected cells and prevention of TNF-α- and interleukin-1β (IL-1β) mediated nuclear factor κB (NF-κB) activation.
Seven transmembrane (7TM) G protein-coupled receptors (GPCRs) are important drug targets. Large DNA viruses (herpes- and pox-) assign large parts of their genomes to exploit GPCRs, however, no such mechanism has yet been described for small viruses (like the paramyxovirinae). The adhesion and barrier-forming receptor ADGRA3 (previously known as GPR125) is widely expressed in the human body with particularly high expression in the choroid plexus. Combined with the subclass B2 defining adhesive properties, this expression pattern is of particular interest, as the choroid plexus, besides being the organ that produces cerebrospinal fluid (CSF), constitutes the main epithelium in the BCSF barrier. The choroid plexus epithelium expresses specific transporter systems that combined with its pinocytotic activity regulate the transchoroidal crossing of substances into the ventricles. We have previously developed a method for choroid plexus explants to study these mechanisms. Several studies have ascribed a role of the BCSF barrier for immune cells and pathogens’ entry into the brain. From the ventricles, the pathogens can elicit inflammatory reactions in the brain, ultimately leading to diseases like meningitis and meningoencephalitis. The transchoroidal crossing into the brain of pathogens can either be 1) transcellular with penetration through the cell, 2) via an opening of tight junctions in a paracellular manner, or 3) through highjacked infected phagocytic host cells (as a “Trojan horse” strategy).
When expressed in cultured cells, we have previously shown that ADGRA3 undergoes constitutive clathrin-mediated, arrestin-independent internalization; however, at present, no G protein-dependent signaling has been described for ADGRA3.
In the VIREX project we have tested the groundbreaking hypothesis that the ADGRA3 (originally known as GPR125) is central for the organ damage caused by mumps virus (MuV) via an interaction with the MuV-encoded SH-protein.
We have studied:
The functional consequences of ADGRA3-SH-interaction at a single cell, organ and whole body level within the context of MuV infection.
The structural requirements for the ADGRA3-MuV interaction using NMR and resolution of crystal structure in preparation for future drug design.
Seven transmembrane (7TM) G protein-coupled receptors (GPCRs) are important drug targets. Large DNA viruses (herpes- and pox-) assign large parts of their genomes to exploit GPCRs, however, no such mechanism has yet been described for small viruses (like the paramyxovirinae). The adhesion and barrier-forming receptor ADGRA3 (previously known as GPR125) is widely expressed in the human body with particularly high expression in the choroid plexus. Combined with the subclass B2 defining adhesive properties, this expression pattern is of particular interest, as the choroid plexus, besides being the organ that produces cerebrospinal fluid (CSF), constitutes the main epithelium in the BCSF barrier. The choroid plexus epithelium expresses specific transporter systems that combined with its pinocytotic activity regulate the transchoroidal crossing of substances into the ventricles. We have previously developed a method for choroid plexus explants to study these mechanisms. Several studies have ascribed a role of the BCSF barrier for immune cells and pathogens’ entry into the brain. From the ventricles, the pathogens can elicit inflammatory reactions in the brain, ultimately leading to diseases like meningitis and meningoencephalitis. The transchoroidal crossing into the brain of pathogens can either be 1) transcellular with penetration through the cell, 2) via an opening of tight junctions in a paracellular manner, or 3) through highjacked infected phagocytic host cells (as a “Trojan horse” strategy).
When expressed in cultured cells, we have previously shown that ADGRA3 undergoes constitutive clathrin-mediated, arrestin-independent internalization; however, at present, no G protein-dependent signaling has been described for ADGRA3.
In the VIREX project we have tested the groundbreaking hypothesis that the ADGRA3 (originally known as GPR125) is central for the organ damage caused by mumps virus (MuV) via an interaction with the MuV-encoded SH-protein.
We have studied:
The functional consequences of ADGRA3-SH-interaction at a single cell, organ and whole body level within the context of MuV infection.
The structural requirements for the ADGRA3-MuV interaction using NMR and resolution of crystal structure in preparation for future drug design.
We found that the eight N-terminal amino acids were essential for the ADGRA3-SH interaction, as only the SH-protein containing this part (1-34), but not the SH-protein (9-57) was able to interact with ADGRA3
We found that the N-terminal SH-protein sequence (1-8aa) is specific for MuV and do not correspond to any human protein sequence suggesting that the SH proteins is indeed a unique ligand and a novel interaction partner for ADGRA3
We detected Adgra3-β-gal expression in the developing brain (E14) with distinct and robust X-gal staining in the choroid plexus. The high Adgra3 expression in the adult brain of C57B2/B6 mice (8-10 weeks of age) was confirmed by RT-qPCR. Intriguingly, the high expression of ADGRA3 in choroid plexus could imply a role for its function of this epithelium and thereby ADGRA3 could be the counterpart to the endothelial expression ADGRA2, which is essential for the blood brain-specific vascularization (10, 11, 26). See findings below where this is exactly what we prove for ADGRA3.
Using an SH-protein, conjugated with a fluorophore (5´TAMRA), we illustrated its binding in the choroid plexus in a dose-dependent manner. The specificity to ADGRA3 was confirmed using Adgra+/+ (wild type C57B2/B6) mice in parallel with Adgra3 knock out (KO) mice (Adgra3cre/cre). These data show that ADGRA3 is robustly expressed at places for MuV-mediated disease and identify the MuV-SH as a novel ligand for ADGRA3. The interaction between the two leads to barrier breakdown, implying a mechanism for MuV entry into organs and highlighting ADGRA3 as a novel target for antiviral treatment.
To gain insight into its role in the choroid plexus, we performed RNA sequencing of mouse choroid plexus after selective elimination of Adgra3 at this site after intraventricular AAV5 shRNA injection. A total of 286 genes significantly increased their RNA expression after silencing of Adgra3 and 51 genes decreased. Functional enrichment analysis of the differentially expressed genes revealed involvement in choroid plexus structure and functional processes for example structure organization, epithelial cell proliferation, cell adhesion and progression of external stimuli to choroid plexus cells. Interestingly, silencing of Adgra3 had a particular impact in the expression of genes involved in the Adgra3 related functions as cell polarity and cell adhesion, including genes of the Cadherin- and Wnt families (Cldh5 and Wnt5a). Moreover, changes were observed in the expression of genes involved in related functions such as ABC transporter (Abca9), extracellular matrix ECM receptor interaction (Vtn), tight junctions (Cldn1), adherent junctions (Ptprb, Lmo7) and focal adhesions (Mylk, Parva).
We found that the N-terminal SH-protein sequence (1-8aa) is specific for MuV and do not correspond to any human protein sequence suggesting that the SH proteins is indeed a unique ligand and a novel interaction partner for ADGRA3
We detected Adgra3-β-gal expression in the developing brain (E14) with distinct and robust X-gal staining in the choroid plexus. The high Adgra3 expression in the adult brain of C57B2/B6 mice (8-10 weeks of age) was confirmed by RT-qPCR. Intriguingly, the high expression of ADGRA3 in choroid plexus could imply a role for its function of this epithelium and thereby ADGRA3 could be the counterpart to the endothelial expression ADGRA2, which is essential for the blood brain-specific vascularization (10, 11, 26). See findings below where this is exactly what we prove for ADGRA3.
Using an SH-protein, conjugated with a fluorophore (5´TAMRA), we illustrated its binding in the choroid plexus in a dose-dependent manner. The specificity to ADGRA3 was confirmed using Adgra+/+ (wild type C57B2/B6) mice in parallel with Adgra3 knock out (KO) mice (Adgra3cre/cre). These data show that ADGRA3 is robustly expressed at places for MuV-mediated disease and identify the MuV-SH as a novel ligand for ADGRA3. The interaction between the two leads to barrier breakdown, implying a mechanism for MuV entry into organs and highlighting ADGRA3 as a novel target for antiviral treatment.
To gain insight into its role in the choroid plexus, we performed RNA sequencing of mouse choroid plexus after selective elimination of Adgra3 at this site after intraventricular AAV5 shRNA injection. A total of 286 genes significantly increased their RNA expression after silencing of Adgra3 and 51 genes decreased. Functional enrichment analysis of the differentially expressed genes revealed involvement in choroid plexus structure and functional processes for example structure organization, epithelial cell proliferation, cell adhesion and progression of external stimuli to choroid plexus cells. Interestingly, silencing of Adgra3 had a particular impact in the expression of genes involved in the Adgra3 related functions as cell polarity and cell adhesion, including genes of the Cadherin- and Wnt families (Cldh5 and Wnt5a). Moreover, changes were observed in the expression of genes involved in related functions such as ABC transporter (Abca9), extracellular matrix ECM receptor interaction (Vtn), tight junctions (Cldn1), adherent junctions (Ptprb, Lmo7) and focal adhesions (Mylk, Parva).
Taken together, we demonstrate in the VIREX project that ADGRA3 is present at sites of MuV manifestations with strong expression in the choroid plexus and that the receptor is important for the barrier function of this organ. We moreover find that the interaction between the SH protein from MuV and ADGRA3 supports virus entry into the brain by influencing the barrier function of ADGRA3. Our study is the first to suggest a host membrane-expressed protein as target to prevent virus entry into the brain. Moreover, a consequence of our findings is that we may be able to take advantage from the knowledge of virus entry into to brain to facilitate transchoroidal drug transport through ADGRA3 targeting with SH-conjugated drugs (patent-application from the University of Copenhagen, priority-date in the end of October 2022).