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Journal of Virology, November 2009, p. 11940-11949, Vol. 83, No. 22
0022-538X/09/$08.00+0     doi:10.1128/JVI.01244-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Kjerstin H. W. Lanke,1 Hilde M. van der Schaar,1 George A. Belov,2 Qian Feng,2,{dagger} Daniël Duijsings,1,{ddagger} Catherine L. Jackson,3 Ellie Ehrenfeld,2 and Frank J. M. van Kuppeveld1*

Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences and Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands,1 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland,2 Laboratoire d'Enzymology et Biochemie Structurales, CNRS, Gif-sur-Yvette, France3

Received 16 June 2009/ Accepted 31 August 2009

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.

* Corresponding author. Mailing address: Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences and Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31-24-3617574. Fax: 31-24-3614666. E-mail: f.vankuppeveld{at}ncmls.ru.nl

{triangledown} Published ahead of print on 9 September 2009.

{dagger} Present address: Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

{ddagger} Present address: Yacht BV, Eindhoven, The Netherlands.

Journal of Virology, November 2009, p. 11940-11949, Vol. 83, No. 22
0022-538X/09/$08.00+0     doi:10.1128/JVI.01244-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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