[Frontiers in Bioscience E2, 143-150, January 1, 2010]

HBV X protein interacts with cytoskeletal signaling proteins through SH3 binding

Huixing Feng1, Tuan Lin Tan1, Dandan Niu1, Wei Ning Chen1

1School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell line and plasmids
3.2. Bacterial expression and protein extraction
3.3. GST purification and pull-down
3.4. Panomics SH3 domain arrays
4. Results
4.1. Proline-rich (PXXP) motif in HBx
4.2. Identification of interacting SH3 domain-containing cellular proteins
4.3. HBx interacts with cytoskeletal and signal transduction proteins
4.4. HBx interaction with cortactin mediated by the proline residues
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

The aim of this study was to investigate interactions between cellular SH3-containing proteins and the proline-rich domain in Hepatitis B Virus (HBV) X protein (HBx) The proline-rich domain of HBx (amino acids 19-58) as well as the relevant site-directed mutagenesis (proline to alanine residues) were cloned into pGEX-5X-1 and expressed as GST-PXXP and GST-AXXA probes. Panomics SH3 domain arrays were probed using both GST-PXXP and GST-AXXA to identify potential interacting SH3 domain containing proteins. The specific interactions were confirmed by the immunoprecipitation of the full-length SH3 domain-containing protein. We report here the binding assay which demonstrated interaction between PXXP domain in HBx and the SH3-domain containing proteins, in particular various signaling proteins involved in cytoskeletal reorganization. Our findings were consistent with similar virus-host interactions via SH3 binding for other viruses such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV) Further characterization of the proline-rich binding to SH3 domains could yield important information for the design of novel therapeutic measures against downstream disease causative effects of HBx in the liver cells.