Molecular Mechanism Of The Influence Of Host Protein ATP6V1A On Rabies Virus Replication

Rabies virus(RABV)infects nearly all kinds of mammals including human beings,and leads the lethal zoonoses-rabies.Rabies causes acute and lethal encephalomyelitis,and almost 100%mortality.Annually,about 55,000 people die from rabies and over 15 million people receive post-exposure prophylaxis worldwide.RABV belongs to the Lyssavirus in the Rhabdoviridae family,and has a nonsegmented negative-strand RNA genome of approximately 12 kb in length.Viral genome consists of five genes,and encodes five viral proteins:the nucleoprotein(N),the phosphoprotein(P),the matrix protein(M),the glycoprotein(G),and the large polymerase protein(L).The M protein of RABV plays crucial roles in viral transcription,replication,assembly,and budding;however,its function during the early stage of virus replication remains unknown.The research mapped the protein interactome between RABV M protein and human host factors.Pull-down assay and mass spectrometry identified a total of 2,672 host proteins that coimmunoprecipitated with the M protein.Functional classification of the associated host proteins indicated that ATP-powered-pumps subunits is a large genre.V-type proton ATPase(V-ATPase)is a type of ATPpowered-pumps,we focused on its catalytic subunit A(ATP6V1 A)because of the importance in providing an acidic environment in the endosomes where RABV first enters.The ATP6V1A protein level was downregulated,upregulated or trans-complemented by small RNA interfering,instant overexpression by plasmids transfecting or transfecting plasmids after interfering by small RNA to research the influence of ATP6V1A on RABV replication;and research the molecular mechanism of interaction of ATP6V1A and M protein by Co-IP and Pull-down assay.In the study,RABV replication was reduced by downregulating the level of ATP6V1A protein.Further research found that knockdown of ATP6V1A inhibited dissociation of M protein and decreased the uncoating of RABV in infected cells;and ATP6V1A co-localized with M protein during viral uncoating.M protein interacted with the whole or middle domain of ATP6V1A(160 aa-309 aa,A160309);this interaction was dependent on the lysine residue at position 256 and the glutamic acid residue at position 279.Interaction of ATP6V1A or A160-309 and RABV M protein was functionally related with the promotion of ATP6V1A on RABV replication.Moreover,transfection of packaged lentivirus established a Vero cell line stably overexpressing ATP6V1A,which support higher level replication of RABV and may provide the potential to improve the production of rabies vaccine.The research screened the associated host proteins of RABV M protein and fucosed on ATP6V1A for further study.ATP6V1A plays an important role in the early stage of RABV replication by promoting virion uncoating and interacting with the M protein.The research promotes understanding of the molecular mechanism of RABV replication especially that associated with M protein,and gain experimental data for the study of anti-viral drug targeting on viral replication mechanism.