Mitochondrial Dysfunction in Lyssavirus-Induced Apoptosis

Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.During coevolution with their hosts, viruses have developed many ways of manipulating the cellular machinery of infected cells. They inhibit or induce apoptosis for their own benefit, with the purpose of increasing viral replication and spread or subverting the host''s immune response (4, 12, 51, 59).Mitochondria have several functions in the cell, including energy production, calcium buffering, and regulation of cellular apoptosis. Death signals in the intrinsic pathway of apoptosis act directly on mitochondria, leading to their dysfunction and the release of proapoptotic factors responsible for the caspase-dependent and/or -independent death pathways (43). The process is tightly regulated positively or negatively by proteins from the Bcl-2 family (32). Caspase activation can be initiated in the extrinsic pathway of apoptosis by death receptors expressed at the cell surface; this later causes mitochondrial dysfunction (8, 20).Lyssaviruses are highly neurotropic viruses associated with rabies, a fatal encephalomyelitis considered to be a reemerging zoonosis throughout most of the world (10). It has been suggested that lyssavirus-induced neuronal apoptosis (1), previously thought to be a principal cause of pathogenesis, is an important defense mechanism against lyssavirus infection (26, 34, 56). However, the molecular basis of lyssavirus-induced neuronal apoptosis is still poorly understood (16, 55). The involvement of the viral glycoprotein (G) in inducing neuronal apoptosis has been extensively shown (13, 38, 39, 45), whereas we have suggested that M is an inducer of neuronal cell death through a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent pathway (29). However, the molecular mechanism of apoptosis has not been precisely defined, and little is known about mitochondrial involvement during lyssavirus infections (46).In this study, we take advantage of the fact that Mokola virus (MOK), a member of the genotype 3 lyssaviruses (5), is known to be less pathogenic than viruses of genotype 1 and, in particular, Thailand virus (THA) (3). We report for the first time the involvement of the mitochondrial machinery during MOK-induced apoptosis. We show that the MOK matrix protein (M-MOK), a previously described apoptogenic factor (29), interacts directly with cytochrome c (cyt-c) oxidase (CcO) subunit I (CcO1), the terminal component of the mitochondrial respiratory chain (MRC). This finding is of interest, as this interaction, which is not found with M-THA, may have a key role in controlling ATP synthesis and cellular respiration during lyssavirus-induced neuronal apoptosis and may contribute to the low pathogenesis of MOK infection.