Rabies virus phosphoprotein interacts with mitochondrial Complex I and induces mitochondrial dysfunction and oxidative stress

Abstract

Our previous studies in an experimental model of rabies showed neuronal process degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion neurons infected with challenge virus standard (CVS)-11 strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases reactive oxygen species (ROS) production and mitochondrial Complex I activity vs. mock infection. We have hypothesized that a RABV protein targets mitochondria and triggers dysfunction. Mitochondrial extracts of mouse neuroblastoma cells were analyzed with a proteomics approach. We have identified peptides belonging to the RABV nucleocapsid protein (N), phosphoprotein (P), and glycoprotein (G), and our data indicate that the extract was most highly enriched with P. P was also detected by immunoblotting in RABV-infected purified mitochondrial extracts and also in Complex I immunoprecipitates from the extracts but not in mock-infected extracts. A plasmid expressing P in cells increased Complex I activity and increased ROS generation, whereas expression of other RABV proteins did not. We have analyzed recombinant plasmids encoding various P gene segments. Expression of a peptide from amino acid 139-172 increased Complex I activity and ROS generation similar to expression of the entire P protein, whereas peptides that did not contain this region did not increase Complex I activity or induce ROS generation. These results indicate that a region of the RABV P interacts with Complex I in mitochondria causing mitochondrial dysfunction, increased generation of ROS, and oxidative stress.

Diabolical effects of rabies encephalitis

“…We transfected the human embryonic cell line HEK-293T (HEK) with plasmids expressing RABV proteins and only the plasmid expressing the RABV phosphoprotein (P) increased Complex I activity and ROS levels, whereas none of the other rabies virus proteins increased Complex I activity or ROS levels (Kammouni et al 2015). We found co-localization of immunofluorescent staining for the RABV P and the voltage-dependent anion channel (VDAC) as a mitochondrial marker (Fig.…”

“…This work strongly suggests that increased Complex I activity explains ROS overproduction, which was supported by Amplex Red assays for hydrogen peroxide production. We now have strong evidence that the RABV P physically interacts with Complex I and increases its activity resulting in increased ROS levels (Kammouni et al 2015).…”

Perspectives in Diagnosis and Treatment of Rabies Viral Encephalitis: Insights from Pathogenesis

“…Kammouni et al [115], using a proteomics approach, demonstrated that the phosphoprotein of rabies virus in in vitro mouse neuroblastoma cell lines causes mitochondrial dysfunction by complexing with the complex I subunit of mitochondria, thereby inducing oxidative stress via induction of reactive oxygen species.…”

Subversion of the Immune Response by Rabies Virus

“…However, cells infected with pathogenic strains do not undergo neuronal apoptosis but are rather affected by neuronal dysfunction, in contrast to the attenuated strains [56,58]. Pathogenic strains of RABV induce neuronal dysfunction, resulting in axonal and dendritic swelling and beading [58,82,83,84,85]. These morphological changes are thought to be sufficient to explain the onset of clinical signs and symptoms [83].…”