Interaction between SARS-CoV helicase and a multifunctional cellular protein (Ddx5) revealed by yeast and mammalian cell two-hybrid systems

Abstract

To reveal the putative cellular factors involved in SARS coronavirus replication, the helicase (Hel, nsp13) of SARS coronavirus was used to screen the cDNA library of rat pulmonary epithelial cells using the yeast two-hybrid system. Positively interacting proteins were further tested using a mammalian cell hybrid system and co-immunoprecipitation in the human A549 cell line, which has been shown to support SARS coronavirus replication. Out of the seven positive clones observed by yeast two-hybrid assay, only the Ddx5 (Asp-Glu-Ala-Asp box polypeptide 5) protein showed specific interaction with SARS-CoV helicase. When expression of DdX5 was knocked down by small interfering RNA (siRNA), SARS coronavirus replication was significantly inhibited in fetal rhesus kidney (FRhK-4) cells. Since Ddx5 is a multifunctional protein that plays important roles in transcriptional regulation, its interaction with SARS coronavirus helicase provides interesting clues for studying virus-host cell interactions in SARS-CoV infections.

Fig. 1

Western blot analysis of SARS-CoV helicase in AH109 cells. pGBKT7-Hel was introduced by transformation into AH109, and the lysates of transformants were transferred to Western blots and probed with GAL4 DNA-BD monoclonal antibodies. The empty vector pGBKT7, encoding a GAL4 BD tag protein, is shown, with a molecular weight of around 20 kDa, while pGBKT7-Hel encoding a GAL4 BD-Hel fusion protein, is shown, with a molecular weight around 87 kDa

Fig. 2

In vivo co-immunoprecipitation of SARS-CoV helicase and cellular protein Ddx5 in A549 cells. a Immunoblotting of protein extracts from a cell line co-expressing HA-Hel with c-Myc-Ddx5, using anti-HA and anti-c-Myc antibodies. b Protein extracts from the cells were first subjected to overnight incubation with anti-HA IgG, and the co-precipitated proteins were detected with anti-c-Myc antibodies. IP and ID refer to the antibodies used for immunoprecipitation, and immunodetection, respectively. HA-Hel and/or Myc-Ddx5 proteins loaded into different lanes are indicated. Only lane 4 was loaded with Hel and Ddx5, which showed co-precipitation of both proteins

Fig. 3

Inhibition of siRNA on the expression of Ddx5 protein (a) and replication of SARS-CoV (b) in FRhK-4 cells. a FRhK-4 cells were transfected with either Ddx5-specifc siRNA (Ddx5-1144, lane 1) or unrelated siRNA (lane 2), cells were disrupted 72 h post-transfection, and whole-cell lysates were examined by Western blotting using anti-Ddx5 antibody. b FRhK-4 cells were transfected with either Ddx5-1144 (group 1) or unrelated siRNA (group 2), and 16–18 h after transfection, the cells were infected with 100 TCID50 of SARS-CoV strain GZ-50. Supernatant was collected 72 h post-infection to measure viral load by real-time RT-PCR and viral titers by back-titration. The relative inhibition rate was compared to those for cells transfected with control siRNA and untreated cells