Structural and Mechanistic Usage and Preference of Human, Dog, and Bat Receptors by Rabies Virus

Abstract Rabies is a lethal zoonotic viral disease causing approximately 59,000 human deaths annually. Recently, several cellular receptors for rabies virus (RABV) entry and internalization have been identified. However, none of these receptors have been demonstrated to be indispensable for RABV entry. Here we describe the RABV receptor preference in vivo , utilizing a replication-competent vesicular stomatitis virus (VSV), in which the VSV surface glycoprotein was replaced with rabies virus glycoprotein. To investigate the specific role of RABV receptors in promoting RABV entry in non-permissive cell line, HaCaT cells were used as a cellular model refractory for RABV infection. Employing virus binding and quantification studies, we demonstrated that ITGB1 and mGluR2 are potential receptors for RABV entry and replication. Consequently, knockout (KO) cell lines corresponding to each of the ITGB1 and mGluR2 receptors were generated using CRISPR/Cas9 mediated knockout. Surprisingly, RABV was still able to enter and replicate in the generated KO cell lines, yet the replication and entry of RABV in KO cells lacking mGluR2 and ITGB1 were significantly reduced; respectively. These findings suggest that RABV utilize these receptors in series rather than sequentially. To test whether RABV utilizes similar receptor preference among human, dog, and bats, the A549, Pa-Br and MDCK cell lines that overexpress receptor orthologs from their respective species were infected with rVSV-dG-RABV-G-GFP and quantified for virus binding and released virus progeny. Our findings revealed that in human cells, ITGB1 increased virus entry, while nAChR enhanced virus replication. In bat cells, ectopic expression of nAChR allowed enhanced virus entry and internalization. While MDCK cells overexpressing ITGB1 enhanced the levels of virus entry and replication. Conclusively, our study, reveals the RABV distinct receptor preference, influenced by the underlying pathways that occur during the interaction between the virus and receptor in different cell lines. Additionally, it emphasizes the significance of host-specific factors in virus entry and replication. Graphical Abstract Author Summary Rabies is a fatal neurological disease, characterized by broad host range and tissue tropism. In accordance with the global goal of eliminating dog-mediated human rabies by 2030, studying the underlying mechanism of RABV entry across distinct species would enable adjustment of RABV control strategies. Owing to RABV wide tropism, multiple cellular receptors have been identified for RABV entry into host cells. Previous studies have proposed that some of RABV receptors could serve as promising candidates for development of antiviral drugs (1). From this perspective, we focused on elucidating RABV receptor preference for viral entry in human, dog, and bat cells. In addition to determining whether RABV utilizes these receptors in parallel or in series which would indicate the potential of the identified RABV cellular receptors as targets for antiviral drugs against rabies. Our results demonstrated varying receptor preference of RABV across species. In addition to revealing that none of RABV receptors solely, govern the broad host range of rabies, suggesting that RABV antiviral drugs targeting host cellular factors may not effectively inhibit RABV entry into cells, while antiviral drugs targeting virus glycoprotein may exhibit greater efficacy. Collectively, our study, contribute to providing mechanistic model for RABV entry in different species.