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 reticulum-to-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.

FIG. 1.

BFA does not affect CVB3 RNA replication in MDCK cells, which contain a BFA-resistant GBF1 protein. (A) Schematic representation of the three BFA-sensitive Arf GEFs. The BFA-resistant mutation in the Sec7 domain of GBF1 in MDCK cells is indicated in boldface. (B) Control MDCK cells and cells treated with 2 μg/ml BFA were stained with antibodies against COP-I or AP1. Note that BFA has no effect on the association of COP-I with Golgi membranes but causes the dissociation of AP1 from the TGN and endosomes in MDCK cells. (C) MDCK cells and HeLa cells were transfected with in vitro RNA transcripts derived from a cDNA encoding a CB3 replicon containing the firefly luciferase gene in place of the P1 capsid coding region. After transfection, the cells were supplied with medium containing BFA or guanidine-HCl, a well known inhibitor of enterovirus RNA replication. Luciferase production was measured at the indicated time points.

FIG. 2.

siRNA-mediated knockdown of GBF1, but not of Arf1, inhibits CVB3 RNA replication in HeLa cells. (A) Cells were treated with siRNA against GBF1 for 40 h and then stained with an antibody against GBF1. The siRNA treatment inhibited GBF1 expression in about 80 to 90% of the cells. (B) Cells were cotransfected with a plasmid encoding EYFP-GBF1 and siRNA against GBF1 (left) or cotransfected with a plasmid encoding Arf1-EYFP and siRNA against Arf1. Fluorescence was monitored at 2 days posttransfection. (C) Cells were treated with siRNAs directed against GBF1 or Arf1 or a scrambled siRNA (control). At 40 h posttransfection, the cells were infected with GFP-expressing CVB3. GFP fluorescence, which is indicative of replication, was monitored at 7 h postinfection. (D) Similar to panel C, but the cells were infected with a Renilla luciferase-expressing CVB3. Luciferase production was measured at 7 h postinfection and is expressed as a percentage of that observed with the no-siRNA control (which was set at 100%). (E) Cells were treated with siRNAs against GBF1 or a scrambled siRNA and then transfected with in vitro transcripts from firefly luciferase-expressing replicons of CVB3 or EMCV. The amount of luciferase produced at 8 h posttransfection was measured and is expressed as a percentage of that observed with the siRNA control (which was set at 100%).

FIG. 3.

GBF1, but not Arfs or Rab1B, rescues CVB3 replication in the presence of BFA. BGM cells were transfected with the indicated GBF1 expression constructs (A), with the indicated Arf1 or Rab1b expression constructs (B), or with combinations of different Arf isoforms (C). All of the expressed proteins were GFP tagged. The next day, the cells were infected with CVB3 at an MOI of 10 and incubated at 37°C in medium with or without BFA. At 8 h (C) or at 8 and 16 h (A and B) postinfection, the cells were lysed by three cycles of freezing and thawing, and the amount of virus was determined by end-point titration. Western blot analysis using an anti-EGFP serum confirmed the expression of the indicated proteins.

FIG. 4.

GBF1-M832L efficiently rescues replication of CVB3 carrying a mutant 3A protein that is impaired in binding GBF1 in the presence of BFA. BGM cells were transfected with EGFP (control) or EYFP-GBF1 M832L. The next day, the cells were infected with either wt CVB3 (A) or CVB3-3A-ins[16]S (B) at an MOI of 10 and incubated at 37°C in medium with or without BFA. Virus titers were determined at 0 h and 8 h postinfection (p.i.). The error bars indicate standard deviations.

FIG. 5.

Differential viral-RNA rescue effects of BFA-resistant GBF1 mutants M832L and A795E in HeLa cells. RNA replication of wt and mutant replicons of PV (A, C, and E) or CVB3 (B, D, and F) in the presence or absence of BFA in nontransfected (control) cells (A and B), in cells transiently transfected with GBF1 M832L (C and D), or in cells transiently transfected with GBF1 A795E. RNA replication was measured by determining the amount of Renilla luciferase production.

FIG. 6.

BFA-resistant GBF1 mutants M832L and A795E differ in their abilities to rescue protein secretion and cell viability in the presence of BFA. (A) HeLa cells were cotransfected with plasmid pCMV-Gluc expressing secreted Gaussia luciferase and either a control plasmid or plasmids expressing GBF1 mutant M832L or A795E. The next day, protein secretion in the presence of 1 μg/ml BFA was monitored. The amount of secreted protein in each sample in the absence of BFA was set at 100%. Western blot analysis (right) showed equal expression of the transfected GBF1 mutant proteins. (B) HeLa cells were transfected with a control plasmid or plasmids expressing GBF1 mutant M832L or A795E, and subsequent cell growth in the presence of the indicated amounts of BFA was monitored by a luminescent cell viability assay. The error bars indicate standard deviations.