Functional Analysis Of Rabies Virus Matrix Protein For Apoptotic Induction,Viral Budding And Microtubule Depolymerization

Rabies virus(RABV),belonging to lyssavirus genus of rhabdoviridae family,is the causative agent of rabies with severely neurological symptoms and almost 100%mortality.Despite degeneration and changed cytoskeletons of neuronal in the brain tissue of RABV-infected cases are detected,the pathogenic mechanism of RABV is still far from clear.In this study,we investigated the relationship between viral M protein and mitochondrial apoptotic pathway,the relationship between viral M protein and microfi lament cytoskeleton during budding process of progeny virus particles,and the effects of RABV infection on microtubule cytoskeleton,in order to supply the foundation for understanding the pathogenic mechanism of RABV and find potentialtargets to develop efficient therapeutic drugs for rabies.Apoptosis is considered as a host innate defense mechanism by eliminating virus-infected cells against rampant viral replication.Throughout the process of pathogen-host co-evolution,viruses have developed many strategies to manipulate infected cell apoptosis.Many viruses inhibit or delay apoptosis to prolong the survival of infected cells and facilitate viral replication and progeny viral production.However,some viruses induce apoptosis to promoting the release and dissemination of viral progeny.In this study,we analyzed the apoptotic pathway triggered RABV infection in the model of mouse neuroblastoma N2a cells infected with the challenge virus standard-11 strain of fixed rabies virus(CVS).Caspases assays and confocal assays showed that RABV infection activated caspase-9 and induced cyto c and AIF released from mitochondria into cytoplasm and nucleus,respectively,suggesting that RABV activates both caspase-dependent and caspase-independent mitochondrial apoptotic pathways to induce apoptosis in the late stages of infection.Expression of viral M protein alone dissipated mitochondrial membrane potential and activated caspae-9 and-3,similar to those described in the context of viral infection.Confocal assay showed that M protein partially colocalized with mitochondria in the cases of both infection and transfection.Construction of site-direct mutants of M and performing confocal and TUNEL assays demonstrated that the a-helix structure(67aa-79aa)of M protein was essential for mitochondrial targeting and induction of apoptosis.Therefore,these results demonstrated that viral M protein directly acts on mitochondria to trigger the mitochondrial apoptotic pathway.Besides,we analyzed the dynamic location of M protein at the cell membrane during the budding process of progeny virus particles.M protein diffusely distributed in the interior side of the cell membrane at 4 hpi,and then accumulated at the cell membrane at 8 hpi.Later,M protein aggregated to form vesicles toward to the outer side of the cell membrane at 12 hpi.Finally,these vesicles were separated from the cell membrane and released into the extracellular,suggesting that progeny virions has completed the process of budding and release.Further analysis provided us a set of close-up,which revealed M protein mediated-budding vesicles colocalized with actin at the cell membrane.Disruption of actin cytoskeleton resulted in viral M protein-mediated budding vesicles detained in the cytoplasm and decrease of virus titer in the cell supernatants.The results showed that M protein-mediated viral budding is dependent on actin cytoskeleton.In addition,we found that RABV infection induced microtubule depolymerization and viral M protein formed filamentous structures by confocal assay.Interestingly,microtubule depolymerization only occurred in those cells where M protein formed filamentous structures.Drugs treatment demonstrated that microtubule depolymerization promoted viral transcription and replication,while microtubule polymerization inhibited viral transcription and replication as well as the formation of filamentous structure of M protein.Furthermore,RABV infection upregulated histone deacetylase 6(HDAC6)expression,resulting in decrease of acetylated a-tubulin.Inhibition of deacetylase activity of HDAC6 notably suppressed viral transcription and replication.In addition,expression of the viral M protein alone significantly induced microtubule depolymerization.These results showed that RABV infection induced microtubule depolymerization to promote viral transcription and replication and that the formation of filamentous structures of viral M protein was closely associated with microtubule depolymerization.Overall,these results indicates that RABV-M protein is a multiple-functional protein,which,on one hand,acts upon mitochondria to induce cell apoptosis.On the other hand,M protein utilizes actin cytoskeleton to promote viral budding on the cell membrane.In addition,M protein is closely related to induction of microtubule depolymerization.