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.

Studying neurotrophin antiviral effect on rabies-infected dorsal root ganglia cultures.

Neurotrophin (NT)-induced modulation of rabies virus adsorption, transcription, and replication were analyzed in adult mouse dorsal root ganglia cultures. Different types of nerve growth factor and NT-3 treatment were tested before infection (pretreatment), during infection (transtreatment) and after withdrawing the viral inoculum (post-treatment). NT pretreatment for 4 days prior to infection produced a significant increase in the quantity of virus adsorbed into cultures and a concomitant increase in genomic viral RNA as measured by real time polymerase chain reaction (PCR). NT pretreatment triggered increased expression of two rabies virus receptors (NCAM and p75NTR); however, no increase in rabies virus transcription and expression could be observed. By contrast, NT treatment during and after infection (trans- and post-treatment) induced a strong decrease in the quantity of viral nucleoprotein genomic and messenger nucleoprotein RNAs. These findings suggested that NT had an intrinsic inhibitory effect on rabies virus infection, which was not counterbalanced by NTs' rabies virus receptor-enhancing property and viral uptake. Adult mouse dorsal root ganglion cultures can be regarded as being a useful model for detecting therapeutic targets and evaluating experimental antiviral drugs.

Lyssavirus matrix protein induces apoptosis by a TRAIL-dependent mechanism involving caspase-8 activation.

Lyssaviruses, which are members of the Rhabdoviridae family, induce apoptosis, which plays an important role in the neuropathogenesis of rabies. However, the mechanisms by which these viruses mediate neuronal apoptosis have not been elucidated. Here we demonstrate that the early induction of apoptosis in a model of lyssavirus-infected neuroblastoma cells involves a TRAIL-dependent pathway requiring the activation of caspase-8 but not of caspase-9 or caspase-10. The activation of caspase-8 results in the activation of caspase-3 and caspase-6, as shown by an increase in the cleavage of the specific caspase substrate in lyssavirus-infected cells. However, neither caspase-1 nor caspase-2 activity was detected during the early phase of infection. Lyssavirus-mediated cell death involves an interaction between TRAIL receptors and TRAIL, as demonstrated by experiments using neutralizing antibodies and soluble decoy TRAIL-R1/R2 receptors. We also demonstrated that the decapsidation and replication of lyssavirus are essential for inducing apoptosis, as supported by UV inactivation, cycloheximide treatment, and the use of bafilomycin A1 to inhibit endosomal acidification. Transfection of cells with the matrix protein induced apoptosis using pathways similar to those described in the context of viral infection. Furthermore, our data suggest that the matrix protein of lyssaviruses plays a major role in the early induction of TRAIL-mediated apoptosis by the release of a soluble, active form of TRAIL. In our model, Fas ligand (CD95L) appears to play a limited role in lyssavirus-mediated neuroblastoma cell death. Similarly, tumor necrosis factor alpha does not appear to play an important role.

Genetic constraints and the adaptive evolution of rabies virus in nature.

We used a molecular evolutionary approach to investigate the species adaptation of rabies virus in nature. A maximum likelihood analysis of selection pressures revealed that the nucleoprotein (N) and glycoprotein (G) genes of natural viral isolates were highly constrained, especially at nonsynonymous sites, in contrast to the higher rates of nonsynonymous evolution observed in viruses subject to laboratory passage. Positive selection was only found at a single amino acid site--position 183 in the ectodomain of the G gene. The low rate of nonsynonymous evolution in natural isolates of rabies virus may be due to constraints imposed by the need to replicate in multiple cell types within the host, which in turn facilitates cross-species transmission, or because viral proteins are not subject to immune selection. Using known dates in the epidemiologic history of European viral isolates, we estimated that overall rates of nucleotide substitution in rabies virus were similar to those observed in other RNA viruses. Assuming that the average rate of synonymous change does not vary among species, we estimated that the current genetic diversity in lyssavirus genotype 1 may have arisen only during the last 500 years.