A Proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication

Abstract

As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, suggesting that the GBF1 environment is linked to viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified multiple proteins previously associated with enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins, many of which normally localize in the nucleus, constituted the largest group, underscoring the massive release of nuclear factors into the cytoplasm of infected cells and their involvement in viral replication. Functional analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly expand the knowledge of the composition of enterovirus replication organelles, provide new insights into viral replication, and offer a novel resource for identifying targets for anti-viral interventions.

The Development of Resistance to an Inhibitor of a Cellular Protein Reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1

“…Interestingly, the depletion of each of these BFA-insensitive ArfGEFs also inhibited the replication of wt replicon, suggesting that BFA-insensitive ArfGEFs (in addition to GBF1) play a currently unrecognized role in the replication process. These data are in line with our recent observation of the recruitment of a BFA-insensitive ArfGEF Cythohesin2/ARNO to the poliovirus replication organelles (65).…”

The development of resistance to an inhibitor of a cellular protein reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1

“…Interestingly, the depletion of each of these BFA-insensitive ArfGEFs also inhibited the replication of wt replicon, suggesting that BFA-insensitive ArfGEFs (in addition to GBF1) play a currently unrecognized role in the replication process. These data are in line with our recent observation of the recruitment of a BFA-insensitive ArfGEF Cythohesin2/ARNO to the poliovirus replication organelles [ 86 ]. It is possible that similar to their normal cellular function, BFA-insensitive ArfGEFs contribute to the activation of Arf6 on the replication organelles.…”