Role of host factors regulating membrane homeostasis in enterovirus replication
Enteroviruses include poliovirus (PV), rhinoviruses (RVs), coxsackieviruses (CVs), and numbered enteroviruses (EVs) that are causative agents of diverse diseases. Besides PV, which causes poliomyelitis, there are more than 280 serotypes of non-polio enteroviruses that can cause various mild and more severe diseases, especially in young children and immunocompromised individuals. Currently, approved antiviral drugs are only available against a few viruses . Despite being one of the largest genera, there are no approved antivirals for treating infections caused by enteroviruses. To target the large variety of enteroviruses as well as to minimize the risk of PV circulation in the post-eradication era, there is a great need for (broad-acting) antivirals against enteroviruses.
Several direct-acting inhibitors have been developed, including capsid binders, which block virus entry, and inhibitors of viral enzymes required for genome replication. Capsid binders and protease inhibitors have been clinically evaluated but failed due to limited efficacy or toxicity issues. As an alternative approach, a function of essential host factors can be a promising target to treat a broad range of enteroviruses. Drug repurposing screens can be useful to uncover promising new inhibitors with disparate viral and host targets. In chapter 2, a detailed summary of antiviral drug development strategies against enterovirus infection as well as the drug development status as of 2016 is described.
The formation of replication organelles (ROs) is a universal feature that can be found in infected cells by any positive-strand RNA viruses including enteroviruses. ROs have a unique structure composed of different membrane lipids, and both viral proteins and host factors are required for the formation of ROs. For enteroviruses, viral protein 3A and the cellular lipid kinase phosphatidylinositol 4-kinase type IIIβ (PI4KB), among other viral and cellular factors, have been suggested to be the key factors that drive this process. In the experimental chapters of this thesis, we provide mechanistic insights on the role of PI4KB and its interactors in enterovirus replication and RO formation.
Single amino acid substitutions in enterovirus 3A proteins were previously demonstrated to confer resistance to inhibitors of PI4KB and oxysterol binding protein (OSBP), which is a lipid transfer protein that controls cholesterol/PI4P exchange and that is essential for enterovirus replication. The exact escape mechanism, however, is not clearly understood.
Although enterovirus replication depends on PI4KB, its role, and that of its product PI4P, is only partially understood. In chapter 3, we employed a mutant coxsackievirus resistant to PI4KB inhibition (i.e., CVB3 3A-H57Y), and uncover that PI4KB activity has distinct functions in RO biogenesis and in proteolytic processing of viral polyprotein. Remarkably, under PI4KB inhibition the mutant virus could replicate its genome in the absence of ROs, using instead the intact Golgi apparatus. This impaired RO biogenesis provided an opportunity to investigate the proposed role of ROs in shielding enteroviral RNA from cellular sensors. Neither accelerated sensing of viral RNA nor enhanced innate immune responses were observed under these conditions. Altogether, these findings point out that enterovirus ROs are not absolutely required for genome replication and question their role as a physical barrier against innate immune sensors.
Growing evidence suggests that alterations in lipid homeostasis affect the proteolytic processing of the enterovirus polyprotein. In chapters 3 and 4, we studied the effect of PI4KB or OSBP inhibition on proteolytic processing of the CVB3 polyprotein during infection as well as in a replication-independent system. The escape mutation (3A-H57Y) rectified a proteolytic processing defect imposed by PI4KB or OSBP inhibition, pointing to a possible escape mechanism. Besides the 3A substitutions in CVB3, another mutation was identified in each of the PI4KB inhibitor-resistant CVB3 pools, namely substitution N2D in 2C. In chapter 4, we show that 2C-N2D by itself did not confer any resistance to inhibitors of PI4KB and OSBP. However, the double mutant (i.e., 2C-N2D/3A-H57Y) showed better replication than the 3A-H57Y single mutant in the presence of inhibitors. We show that both PI4KB and OSBP inhibitors specifically affected the cleavage at the 3A-3B junction, and that mutation 3A-H57Y recovered impaired proteolytic processing at this junction. Although 2C-N2D enhanced replication of the 3A-H57Y single mutant, we did not detect additional effects of this substitution on polyprotein processing, which leaves the mechanism of how 2C-N2D contributes to the resistance to be revealed.
The enteroviral 3A protein recruits PI4KB to ROs, but the exact mechanism remained elusive. Chapters 5 and 6 describe how viral 3A protein recruits PI4KB via another host factor, Acyl-coenzyme A binding domain containing 3 (ACBD3). In chapter 5, we investigated the role of ACBD3 in PI4KB recruitment to ROs during enterovirus infection using ACBD3 knockout cells. Replication of representative enteroviruses and rhinoviruses was all impaired in ACBD3 knockout cells and PI4KB recruitment by different enterovirus 3A proteins was not observed as well. In the absence of ACBD3, individually expressed 3A was found in the ER instead of the Golgi, indicating that ACBD3 is required for proper localization of 3A. Reconstitution of wild-type ACBD3 restored both PI4KB recruitment and 3A localization, while an ACBD3 mutant that cannot bind to PI4KB restored only 3A localization, but not virus replication. Consistently, reconstitution of a PI4KB mutant that cannot bind ACBD3 failed to restore virus replication in PI4KB knockout cells. By utilizing ACBD3 mutants lacking specific domains, we show that Acyl-coenzyme A binding (ACB) and charged amino acids region (CAR) domains are dispensable for 3A-mediated PI4KB recruitment and efficient enterovirus replication. Both the glutamine-rich region (Q) and the Golgi dynamics domain (GOLD) that are mediating interaction with PI4KB and 3A, respectively, are required for PI4KB recruitment and efficient enterovirus replication. Altogether, our data provide new insight into the central role of ACBD3 in recruiting PI4KB by enterovirus 3A and reveal the minimal domains of ACBD3 involved in recruiting PI4KB and supporting enterovirus replication.
In chapter 6, we determined the crystal structure of the ACBD3 GOLD domain together with 3A proteins from several enteroviruses (PV, EV-A71, EV-D68, and RV-B14) to gain a better understanding of structural determinants of ACBD3 recruitment to the viral replication sites. In addition to the characterization of multiple 3A:ACBD3 GOLD complex, we also provide evidence supporting the presence of ACBD3-3A heterotetramers, which is induced by 3A-3A interaction. A model of ACBD3 GOLD:3A complex on the lipid bilayer was generated to understand how the complex and membranes would interact. By utilizing mutants of 3A or ACBD3 that are generated based on structural information, we identified the critical binding interface mediating 3A-ACBD3 interaction as well as 3A-3A dimerization and ACBD3-membrane interaction. Superposition of the crystal structures of the enterovirus 3A:GOLD complexes and previously identified kobuvirus 3A:GOLD complexes revealed that the enterovirus and kobuvirus 3A proteins bind to the same regions of the ACBD3 GOLD domain. In a nutshell, we uncovered a striking convergence in the mechanisms of how distinct picornaviruses recruit ACBD3 and its downstream effectors to the replication sites.
Protein c10orf76, a PI4KB-interacting protein that is poorly characterized, has been identified as a host factor required for coxsackievirus A10 (CVA10). In chapter 7, we aimed to identify the function of c10orf76 in uninfected cells as well as its possible role in enterovirus replication. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and revealed that binding is mediated by the kinase linker of PI4KB. Complex-disrupting mutations demonstrated that PI4KB is required for the recruitment of c10orf76 to the Golgi, while c10orf76 did not affect the localization of PI4KB. An intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses, CVA10 and poliovirus. Intriguingly, c10orf76 also contributed to the localization of GBF1 to the Golgi and subsequent activation of Arf1 at the Golgi. These findings provide a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi and at the virus replication sites.