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  current news   Press   selected story    
     
  25th January 2010  
 

The hepatitis C virus core protein contains a BH3 domain that regulates apoptosis through specific interaction with human Mcl-1.

 
 




Author
Mohd-Ismail NK, Deng L, Sukumaran SK, Yu VC, Hotta H, Tan YJ.

Collaborative Anti-Viral Research Group, Institute of Molecular and Cell Biology, Singapore.

Abstract
The hepatitis C virus (HCV) core protein is known to modulate apoptosis and contribute to viral replication and pathogenesis. In this study, we have identified a Bcl-2 homology 3 (BH3) domain in the core protein that is essential for its proapoptotic property. Coimmunoprecipitation experiments showed that the core protein interacts specifically with the human myeloid cell factor 1 (Mcl-1), a prosurvival member of the Bcl-2 family, but not with other prosurvival members (Bcl-X(L) and Bcl-w). Moreover, the overexpression of Mcl-1 protects against core-induced apoptosis. By using peptide mimetics, core was found to release cytochrome c from isolated mitochondria when complemented with Bad. Thus, core is a bona fide BH3-only protein having properties similar to those of Noxa, a BH3-only member of the Bcl-2 family that binds preferentially to Mcl-1. There are three critical hydrophobic residues in the BH3 domain of the core protein, and they are essential for the proapoptotic property of the core protein. Furthermore, the genotype 1b core protein is more effective than the genotype 2a core protein in inducing apoptosis due to a single-amino-acid difference at one of these hydrophobic residues (residue 119). Replacing this residue in the J6/JFH-1 infectious clone (genotype 2a) with the corresponding amino acid in the genotype 1b core protein produced a mutant virus, J6/JFH-1(V119L), which induced significantly higher levels of apoptosis in the infected cells than the parental J6/JFH-1 virus. Furthermore, the core protein of J6/JFH-1(V119L), but not that of J6/JFH-1, interacted with Mcl-1 in virus-infected cells. Taken together, the core protein is a novel BH3-only viral homologue that contributes to the induction of apoptosis during HCV infection.

 

 
 

 
 


Figure Legend: Comparison of parental J6/JFH-1 and mutant J6/JFH-1(V119L) recombinant viruses. Huh7.5 cells were infected with recombinant HCV at a multiplicity of infection of 0.1 CIU/cell or with a mock preparation, and various assays were performed at different days after infection. (A) Cell viabilities were determined. (B) Caspase-3 activity per cell was determined. (C) The amount of DNA fragmentation per cell was determined. (D) The production of cell-free infectious virus particles was determined. (E) Virus spread in the culture was quantitated. (F) HCV RNA replication was determined by quantitative real-time PCR analysis. (G) Interaction of the core protein with Mcl-1 was determined by coimmunoprecipitation experiments at 3 days p.i. IP was performed using anti-Mcl-1 or anti-HA rabbit polyclonal antibodies and protein A agarose beads. The amounts of the core protein in the lysates before (lanes 1 and 2) and after IP (lanes 3 to 6) were determined by Western blot analysis with an anti-core monoclonal antibody (top). Similarly, the amounts of endogenous Mcl-1 in the samples were determined using an anti-Mcl-1 monoclonal antibody (bottom). Statistical analysis was performed using the one-way analysis of variance to determine if the differences between parental and mutant viruses were statistically significant, and those with P values of <0.05 (marked by asterisks) are considered statistically significant. Data were obtained from three independent experiments, each with triplicate cultures.

Published in J Virol. 2009 Oct;83(19):9993-10006.

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