Proteomic and phosphoproteomic analysis of rabies pathogenesis in the clinical canine brain and identification of a kinase inhibitor as a potential repurposed antiviral agent

Abstract

Rabies is a fatal zoonosis caused by the rabies virus (RABV) that has afflicted humans for thousands of years. RABV infection leads to neurological symptoms and death; however, its pathogenesis in the brain is unclear, which complicates patient care. Given that no treatment exists for symptomatic cases, there is an urgent need for effective antiviral drugs. In this study, we aimed to investigate the pathogenic mechanism of RABV in the brain and screen for potential anti-RABV drugs. Protein samples were extracted from the brains of RABV-positive and RABV-negative dogs, and proteomic and phosphoproteomic analyses were conducted. The results showed that the synaptic vesicle cycle is critical to RABV pathogenesis. The kinases involved in the phosphorylation of proteins in the synaptic vesicle cycle were identified and examined as potential drug targets. Casein kinase 2 and protein kinase C were found to be key kinases for RABV replication, and five inhibitors of these enzymes were tested for their anti-RABV properties. Pretreating cells with the kinase inhibitor sunitinib significantly reduced the viral yield after RABV infection. Our findings suggest that RABV interferes with synaptic communication, which leads to rabies, and that inhibiting a vital kinase can reduce viral production. Hence, our findings have implications for the development of rabies treatment regimes.

Fig 1. Gene ontology analysis results for the proteomic and phosphoproteomic data.

A. Results obtained using the proteomic data. B. Results obtained using the phosphoproteomic data.

Fig 2. The analysis of protein-protein interaction network from proteomic results.

The results of the STRING analysis performed with the differentially expressed proteins. The red circles indicate proteins involved in the synaptic vesicle cycle.

Fig 3. The differentially expressed and phosphorylated proteins associated with the synaptic vesicle cycle.

The synaptic vesicle cycle, adapted from the Kyoto Encyclopedia of Genes and Genomes database (pathway entry: cfa04721), and its five steps: docking (1), priming (2), fusion (3), full fusion (4), and endocytosis (5). The red rectangles indicate differentially expressed proteins, and the blue rectangles indicate differentially phosphorylated proteins.

Fig 4. Identification of kinases involved in the phosphorylation of RABV phosphoprotein, synaptotagmin-1, and syntaxin-binding protein 1.

A. Schematic diagram showing the phosphorylation sites and their corresponding kinases. B. Pie chart showing the percentage of phosphorylation sites on the target proteins associated with each kinase.

Fig 5. Rabies virus production in kinase inhibitor-treated Neuro-2a cells at three time points.

Data are shown as mean ± standard error. * p < 0.05. hpi: hours post-infection.