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

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. ### Competing Interest Statement The authors have declared no competing interest.