GBF1- and ACBD3-Independent Recruitment of PI4KIIIβ to Replication Sites by Rhinovirus 3A Proteins

Abstract

c PI4KIII␤ recruitment to Golgi membranes relies on GBF1/Arf and ACBD3. Enteroviruses such as poliovirus and coxsackievirus recruit PI4KIII␤ to their replication sites via their 3A proteins. Here, we show that human rhinovirus (HRV) 3A also recruited PI4KIII␤ to replication sites. Unlike other enterovirus 3A proteins, HRV 3A failed to bind GBF1. Although HRV 3A was previously shown to interact with ACBD3, our data suggest that PI4KIII␤ recruitment occurred independently of both GBF1 and ACBD3. E nteroviruses (family Picornaviridae), such as poliovirus (PV) and coxsackievirus B3 (CVB3), rely on host factor phosphatidylinositol-4-kinase III␤ (PI4KIII␤) for genome replication (1, 2). The recruitment of PI4KIII␤ to the replication sites is mediated by the 3A viral nonstructural protein (1). PI4KIII␤ is normally recruited to the Golgi membranes by the small GTPase Arf1 (3) and the Arf1 activator guanine nucleotide exchange factor (GEF) GBF1 (4). ACBD3 (acyl-coenzyme A [CoA]-binding protein domain 3) also interacts with PI4KIII␤ to recruit it to the Golgi membranes (5, 6). CVB3 3A and PV 3A both interact with the N terminus of GBF1 (7, 8) as well as ACBD3 (9). However, we and others recently showed that the PI4KIII␤ recruitment by 3A of PV and CVB3 seems to occur independently of both GBF1/Arf1 and ACBD3 (9, 10). We studied this by individually expressing mutant 3A proteins of CVB3 that no longer interact with GBF1 (9). Unfortunately, these 3A mutations render CVB3 unviable (F. J. M. van Kuppeveld, unpublished data); thus, we have not yet been able to study the role of GBF1 in PI4KIII␤ recruitment in infected cells. Yeast two-hybrid analysis suggests that the 3A proteins of human rhinoviruses (HRV), which also belong to the Enterovirus genus, do not interact with the N terminus of GBF1 (8). In this study, we set out to investigate the consequences of the inability of HRV 3A proteins to interact with GBF1, focusing on the possible role of GBF1 and ACBD3 in PI4KIII␤ recruitment and virus replication.First, we studied the interaction between the N terminus of GBF1 and the 3A proteins of HRV2 and HRV14, which are members of group A and B rhinoviruses, respectively, in a mammalian environment, employing the mammalian two-hybrid (M2H) assay (Promega) as described previously (9). We used the 3As of CVB3, PV, and mengovirus, belonging to the Cardiovirus genus of the Picornaviridae, as controls. Expression of all 3A proteins was verified by Western blot analysis (Fig. 1A, lower half). Importantly, all enterovirus 3A proteins were previously tested and validated to be competent in interacting with other proteins in the M2H assay (9). Both CVB3 3A and PV 3A interacted with the N terminus of GBF1, while no interaction was detected for mengovirus 3A (Fig. 1A, upper panel). HRV2 3A did not bind the N terminus of GBF1, while HRV14 3A interacted to only a limited extent. We considered the possibility that the HRV 3A proteins bind to the C terminus of GBF1. Therefore, we studied whether 3A expression induced recruitment of full-length...

Phosphatidylinositol 4-kinases: Function, structure, and inhibition

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