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

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

Interaction of Poliovirus Capsid Proteins with the Cellular Autophagy Pathway

The capsid precursor P1 constitutes the N-terminal part of the enterovirus polyprotein. It is processed into VP0, VP3, and VP1 by the viral proteases, and VP0 is cleaved autocatalytically into VP4 and VP2. We observed that poliovirus VP0 is recognized by an antibody against a cellular autophagy protein, LC3A. The LC3A-like epitope overlapped the VP4/VP2 cleavage site. Individually expressed VP0-EGFP and P1 strongly colocalized with a marker of selective autophagy, p62/SQSTM1. To assess the role of capsid proteins in autophagy development we infected different cells with poliovirus or encapsidated polio replicon coding for only the replication proteins. We analyzed the processing of LC3B and p62/SQSTM1, markers of the initiation and completion of the autophagy pathway and investigated the association of the viral antigens with these autophagy proteins in infected cells. We observed cell-type-specific development of autophagy upon infection and found that only the virion signal strongly colocalized with p62/SQSTM1 early in infection. Collectively, our data suggest that activation of autophagy is not required for replication, and that capsid proteins contain determinants targeting them to p62/SQSTM1-dependent sequestration. Such a strategy may control the level of capsid proteins so that viral RNAs are not removed from the replication/translation pool prematurely

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.

The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics

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

Partial funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.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.https://doi.org/10.1371/journal.ppat.101090