The ATPase ATP6V1A facilitates rabies virus replication by promoting virion uncoating and interacting with the viral matrix protein

Abstract

Rabies virus (RABV) matrix protein (M) plays crucial roles in viral transcription, replication, assembly, and budding; however, its function during the early stage of virus replication remains unknown. Here, we mapped the protein interactome between RABV M and human host factors using a proteomic approach, finding a link to the V-type proton ATPase (V-ATPase) catalytic subunit A (ATP6V1A) which is located in the endosomes where RABV first enters. By downregulating or upregulating ATP6V1A expression in HEK293T cells, we found that ATP6V1A facilitated RABV replication. We further found that ATP6V1A was involved in the dissociation of incoming viral M proteins during viral uncoating. Co-immunoprecipitation demonstrated that M interacted with the full length or middle domain of ATP6V1A, which was dependent on the lysine residue at position 256 and the glutamic acid residue at position 279. RABV growth and uncoating in ATP6V1A-depleted cells was restored by trans-complementation with the full length or interaction domain of ATP6V1A. Moreover, stably overexpressed ATP6V1A enhanced RABV growth in Vero cells which are used for the production of rabies vaccine. Our findings identify a new partner for RABV M proteins and establish a new role of ATP6V1A by promoting virion uncoating during RABV replication.

Rabies Infection: An Overview of Lyssavirus-Host Protein Interactions

“…Analysis of this PIN demonstrated seven targeted signaling pathways, including WNT, MAPK/ERK, RAS, PI3K/AKT, toll-like receptor, JAK/STAT, and NOTCH, were involved in controlling cell cycle, cell survival, viral replication and folding, synapse regulation, and regulation of immunity. Phospholipase [25] , Binding inhibition assay [26] G RABV (CVS strain) NCAM Binding inhibition assay, Virus neutralization [27] G RABV (street strain) RABV (field strains, CVS strain & PV strain), EBLV-2 p75NTR Screening of the cDNA library, Co-IP [28] Reverse binding assay [29] G RABV (ERA strain, CVS-24 strain & street virus GX/09), WCBV mGluR2 RNAi strategy, Co-IP, Pull-down assay [35] G RABV (Virulent strains) MAST1,2 Yeast-two hybrid assay [108] G RABV (Attenuated strains) PTPN4 MAST2 DLG2 MPDZ Yeast-two hybrid assay [108] G RABV (CVS-11, SAD strains) SNAP25 Colocalization, Co-IP [37] N RABV (CVS strain) HSP70 1A,1B Immunoaffinity column immobilized with anti-N [39] N RABV (HEP-Flury strain) (CCTγ) Colocalization, RNAi strategy [42] N RABV (HEP-Flury strain) PFDN1 Colocalization [43] L RABV (SAD B19 strain) DLC1 Colocalization, Mutation in DLC1 motif [47] M RABV NEDD4 GST fusion proteins, Far-western blot assay [95] M RABV YAP1 Far-western blot assay [95] M RABV (PV strain) eIF3h Yeast-two hybrid assay, Surface plasmon resonance [92] M MOKV Cco1 Yeast-two hybrid assay, Co-IP, Colocalization [93] M RABV (Thailand, SAD B19, PV strains), MOKV, LBV, EBLV-1) RelAp43 Yeast-two hybrid assay, Co-IP [94] M RABV (a street strain) JAK1 Protein complementation assay [55] M RABV (a street strain) STAT1 Protein complementation assay [55] M RABV (ERA strain) ATP6V1A Co-IP, Pull-down assay [99] P RABV (CVS-11 strain), MOKV DLC1,2 Yeast-two hybrid assay [79] , Co-IP [78,79] , VirHostNet Database P, P3 isoform RABV (CVS strain) PML Co-IP, Colocalization [50] P RABV (SAD l16, CVS, SHBRV strains), MOKV, ABLV STAT1,2 Yeast-two hybrid assay [51] , Co-IP [51,104] ,VirHostNet database P RABV (CVs strain) STAT3 Co-IP, Colocalization…”

“…They also suggested that M-STAT1 interaction induces the cytoplasmic retention of STAT1, therefore, enhances the capacity of RABV P to bind STAT1 and interferes with the downstream events in the pathway [ 55 ] . Just recently, a new function for M has been discovered in virion uncoating via binding to V-ATPase catalytic subunit A (ATP6V1A) [ 99 ] . V-ATPase complex is involved in endosomal acidification by pumping H + from the cell cytoplasm into the lumen.…”

“…Overexpression and knockout of M partner showed increased and suppressed replication of RABV, respectively. The role of ATP6V1A in RABV uncoating, which in fact facilitates the virus replication, has also been reported [ 99 ] . After RABV entry into the host cell, the endosome-containing virus becomes acidic, and the conformation of RABV G is changed to stimulate virus-endosome membrane fusion.…”

“…Lastly, the involvement of ATP6V1A, exactly in dissociation and separation of RABV M from nucleocapsids, has been demonstrated. The precise molecular mechanism of RABV M separation from nucleocapsids remains to be cleared [ 99 ] . A diagram of the explained M-host PPIs is depicted in Figure 5 .…”

Function of Host Protein Staufen1 in Rabies Virus Replication

“…Rabies is an important zoonotic infectious disease caused by rabies virus (RABV), with a mortality rate of almost 100% [ 1 ]. Neurotropic RABV causes fatal encephalomyelitis after infection of the central nervous system, and approximately 60,000 people die from rabies worldwide each year, with more than 15 million people receiving post-exposure prophylaxis [ 2 , 3 ].…”