GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for Coxsackievirus B3 RNA Replication

Abstract

The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulumto-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

“…CVB3 and CVB3-Rluc, which contains the Renilla luciferase gene upstream of the capsid coding region, were obtained by transfection of RNA transcripts derived from the full length infectious clones p53CB3/T7 and pRLuc-53CB3/T7 in BGM cells as described before [21,62]. CVB3(-Rluc) carrying the V45A or H57Y mutation in the 3A protein were obtained in a similar manner using plasmids p53CB3/T7-3A-V45A or -H57Y described elsewhere [12] or pRluc-53CB3/T7-3A-V45A or -H57Y.…”

Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

“…In order to clarify this, we compared the effects of PIK93 on the replication of WT and replication-deficient FMDV replicons with those on the replication of CVB3, previously shown to be dependent on PI4K activity (Lanke et al, 2009). Because of the different reporters used, replication was monitored by luciferase assay or by GFP expression.…”

“…A GFP reporter was inserted into domain III of the NS5A protein; this insertion was tolerated such that the sub-genomic replicon retained WT replicative ability, as described previously (Moradpour et al, 2004). A CVB3 sub-genomic replicon, pRib-Fluc-CB3/T7, was also employed, and a replication-deficient mutant (pRib-Fluc-CB3/T7-3A) was generated by alanine substitution of residues 6-10, preventing binding of 3A to host-protein GBF1 (Lanke et al, 2009). …”

“…There are two types of well-defined families of PI4Ks, type II (PI4KIIa and PI4KIIb) and type III (PI4KIIIa and PI4KIIIb). Previous studies on PV and CVB3 have identified PI4KIIIb as the host enzyme upregulated in viral replication factories (Arita et al, 2011;Belov et al, 2007;Hsu et al, 2010;Lanke et al, 2009). Depletion of PI4KIIIb activity within infected cells by RNA silencing or the use of kinase inhibitors, such as PIK93 (Knight et al, 2006), appeared to block PV and CVB3 viral RNA synthesis and virus replication.…”

“…Replicons have also been used to study the replication of a number of other viruses. Here, we have also used hepatitis C virus (HCV) sub-genomic replicons pSGR-Luc-GFP-JFH-1 (Jones et al, 2007) and SGR-feo-JFH-1 (Wyles et al, 2009), together with a Coxsackievirus B3 (CVB3) sub-genomic replicon, pRib-Fluc-CB3/T7 (Lanke et al, 2009). …”