Identification and functional characterization of the GBF1-controlled network of host proteins supporting enterovirus replication

Abstract

The genus Enterovirus of the Picornaviridae family contains many established and emerging pathogens. However, licensed vaccines are currently available only against poliovirus and enterovirus A71. No therapeutics have been officially approved to treat any enterovirus infections, although some are being developed. To find suitable targets for antivirals and control the infections, we need to understand the virus's life cycle better and identify the cellular factors involved in virus infection. Enterovirus genome replication occurs on the unique membranes known as replication organelles (ROs). A Golgi resident protein, GBF1, is recruited to the ROs by a viral protein 3A. GBF1 activates small GTPases Arf, which are critical regulators of the cellular secretory pathway. Here, we investigated the mechanistic details of GBF1-dependent Arf activation during enterovirus replication and characterized the proteome of the ROs in the vicinity of GBF1. We showed that Arf1 appeared to be the first to associate with the ROs, followed by other Arfs. Once activated and recruited to the ROs, all Arfs except Arf3 were no longer sensitive to inhibition of GBF1, suggesting that they do not actively cycle between GTP- and GDP-bound states in infected cells. siRNA depletion studies demonstrated an increased sensitivity of polio replication to inhibition of GBF1 in Arf1-, and to a lesser extent, Arf6-depleted cells, indicating the importance of GBF1-mediated activation of these Arfs for the viral replication. Taking advantage of the GBF1 recruitment to the ROs and GBF1’s essential role in enterovirus replication, we used a GBF1 construct fused to APEX2 peroxidase to explore the proteome of the ROs by proximity biotinylation. Among the proteins biotinylated in infected cells were the known cellular factors recruited to the ROs, including PI4KIII, OSBP, and ACBD3, indicating that these proteins are localized close to GBF1. Among the viral proteins, the intermediate products of the polyprotein processing were overrepresented, suggesting that GBF1 is localized close to the sites of active polyprotein processing. About 85% of the proteins identified by MS have not been previously associated with enterovirus infection. Gene ontology analysis revealed a significant enrichment of RNA binding and mRNA metabolic processes, suggesting a close localization of GBF1 to the RNA replication complexes. siRNA knockdown functional analysis of the selected proteins showed the recruitment of both proviral and antiviral factors to the ROs. Collectively, our work revealed important details about the involvement of Arfs in the replication process, introduced a highly efficient system to investigate the proteome of the enterovirus ROs, and provided novel data about the protein composition of the GBF1-enriched environment in the replication sites.