Bhaskar Barnwal1,2, Helen Karlberg3, Ali Mirazimi3,4,5, Yee-Joo Tan1,2,6
1 Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University
Health System (NUHS), National University of Singapore
2 Institute of Molecular and Cell Biology,
A*STAR (Agency for Science, Technology and Research), Singapore
3 Public Health agency of Sweden,
SE-171 82 Sweden
4 Karolinska Institute, Stockholm, SE-171 77, Sweden
5 National Veterinary Institute, SE- 756 51 Uppsala.
6 To whom correspondence should be addressed: Yee-Joo Tan, MD4, 5 Science Drive 2, Singapore 117597;
Tel: +65-65163692; Fax: +65-67766872; E-mail: Yee_Joo_TAN@NUHS.edu.sg
Published in Biological Chemistry on 16 November 2015.
Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study for the first time suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid (aa) residues, in the CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by the caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase (PARP), in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93-140 aa residues. Using alanine scanning, we demonstrated that L127 and L135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that L127 and L135 are important residues for the disruption of mitochondrial membrane potential by NSs. Thus, these results indicate that the C-terminal of CCHFV NSs triggers mitochondrial membrane permeabilization leading to activation of caspases which ultimately leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells.
NSs disrupts the mitochondrial membrane potential in HeLa cells. (A) The subcellular localization of transiently transfected DN-GRIM19-HA was detected by anti-HA antibody and Alexa-Fluor-488-conjugated secondary antibody (green). The subcellular localization of transiently transfected (B) myc-NSs and (C) myc-NSs L127A and L135A were detected by anti-myc antibody and Alexa-Fluor-488-conjugated secondary antibody (green). Mitochondria were labeled with MitoTracker Red CMXRos (red) and nuclei were counterstained with DAPI (blue). At least three independent experiments were performed with a representative set of images shown here.
For more information on Yee Joo TAN's lab, please click here.