ACBD3 is an essential pan-enterovirus host factor that mediates the interaction between viral 3A protein and cellular protein PI4KB

Abstract

250 words 16Total word counts: 4166 words 17. CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/375550 doi: bioRxiv preprint first posted online Jul. 26, 2018; Abstract 18The enterovirus genus of the picornavirus family includes a large number of important human pathogens 19 such as poliovirus, coxsackievirus, enterovirus-A71, and rhinoviruses. Like all other positive-strand RNA 20 viruses, genome replication of enteroviruses occurs on rearranged membranous structures called replication 21 organelles (ROs). Phosphatidylinositol 4-kinase IIIβ (PI4KB) is required by all enteroviruses for RO 22 formation. The enteroviral 3A protein recruits PI4KB to ROs, but the exact mechanism remains elusive. 23Here, we investigated the role of Acyl-coenzyme A binding domain containing 3 (ACBD3) in PI4KB 24 recruitment upon enterovirus replication using ACBD3-knockout (ACBD3 KO ) cells. ACBD3 knockout 25 impaired replication of representative viruses from four enterovirus and two rhinovirus species. PI4KB 26 recruitment was not observed in the absence of ACBD3. The lack of ACBD3 also affected the localization 27 of individually expressed 3A, causing 3A to localize to the endoplasmic reticulum instead of the Golgi. 28Reconstitution of wt ACBD3 restored PI4KB recruitment and 3A localization, while an ACBD3 mutant 29 that cannot bind to PI4KB restored 3A localization, but not virus replication. Consistently, reconstitution 30 of a PI4KB mutant that cannot bind ACBD3 failed to restore virus replication in PI4KB KO cells. Finally, 31 by reconstituting ACBD3 mutants lacking specific domains in ACBD3 KO cells, we show that Acyl-32 coenzyme A binding (ACB) and charged amino acids region (CAR) domains are dispensable for 3A-33 mediated PI4KB recruitment and efficient enterovirus replication. Altogether, our data provide new insight 34 into the central role of ACBD3 in recruiting PI4KB by enterovirus 3A and reveal the minimal domains of 35 ACBD3 involved in recruiting PI4KB and supporting enterovirus replication. 36 37. CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/375550 doi: bioRxiv preprint first posted online Jul. 26, 2018; Importance 38As all other RNA viruses, enteroviruses reorganize host cellular membranes for efficient genome 39 replication. A host lipid kinase, PI4KB, plays an important role on this membrane rearrangement. The exact 40 mechanism of how enteroviruses recruit PI4KB was unclear. Here, we revealed a role of a Golgi-residing 41 protein, ACBD3, as a mediator of PI4KB recruitment upon enterovirus replication. ACBD3 is responsible 42 for proper localization of enteroviral 3A proteins in host cells w...

Essential domains of phosphatidylinositol‐4 kinase III β required for enterovirus replication

“…These findings indicate that interaction of PI4KB with these host factors may affect expression of PI4KB, but is not essential for EV replication once PI4KB has been expressed. The interaction of ACBD3 with PI4KB seems essential for the function of ACBD3 in EV replication and in PI4KB activity in an ACBD3‐knockout cell line; however, our results suggest that direct regulation of PI4KB activity is not the essential role of ACBD3 in EV replication. Our findings further define the role of ACBD3 and other PI4KB‐binding proteins in EV replication and suggest that PI4KB is functionally independent of these host proteins.…”

“…Knockdown of ACBD3 by small interfering RNA does not affect PV or CVB3 replication, suggesting that viral genus/species specific role of ACBD3. Knockout of ACBD3 can suppress EV infection about 10‐ to 100‐fold depending on the viral species . Recent structural analysis has revealed the modes of binding of ACBD3 to viral 3A protein and to PI4KB .…”

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

“…c10orf76 is highly conserved in vertebrates and we find a strong correlation between the conservation of the kinase linker region of PI4KB and the PI4KB-binding site in c10orf76, suggesting that a key role of c10orf76 is linked to its ability to form a complex with PI4KB. One of the most well-conserved 3A binding partners in enteroviruses is the Golgi resident protein ACBD3, which interacts with the central part of 3A and recruits as well as activates PI4KB [11,13,18,29,31]. In vitro, c10orf76 led to decreased lipid kinase activity of PI4KB.…”

“…Specifically, while Coxsackievirus A10 (CVA10) replication was impaired in c10orf76 knockout cells, the replication of CVB1 was not. Due to the notoriously difficult nature of transfecting HAP1 cells, we determined the importance of the c10orf76-PI4KB interaction for virus replication in HeLa PI4KB knockout cells transfected with different PI4KB expression plasmids as previously described [29]. We set out to investigate the importance of the c10orf76-PI4KB interaction for replication of CVA10 and PV1.…”

“…The 3A proteins from enteroviruses (i.e., Poliovirus, Rhinovirus, Coxsackievirus, Rhinovirus, and Enterovirus 71) and Aichivirus recruit PI4KB to replication organelles through an interaction with ACBD3 [11,13,[16][17][18][29][30][31][32]. A key component of manipulating PI4KB to generate PI4P-enriched replication organelles is the interaction of viral 3A proteins with host PI4KBbinding proteins.…”