The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis

Bhaskar Barnwal,Helen Karlberg,Ali Mirazimi,Yee-Joo Tan

doi:10.1074/jbc.m115.667436

Abstract

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 residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, 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 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 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 Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus 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.

Highlights

Crimean-Congo hemorrhagic fever virus (CCHFV),2 an arthropod-borne virus, is a member of the genus Nairovirus in the family Bunyaviridae [1]
Disruption of the mitochondrial membrane potential by the CCHFV NSs protein is dependent on these key residues
NSs Expression in CCHFV-infected Cells—The CCHFV NSs protein is composed of 150 amino acid residues, with its secondary structure predicted by PSIPRED as shown in Fig. 1 [27]

Summary

Introduction

Crimean-Congo hemorrhagic fever virus (CCHFV), an arthropod-borne virus, is a member of the genus Nairovirus in the family Bunyaviridae [1]. La Crosse encephalitis virus NSs has been shown to induce caspase activation and apoptosis [7, 8]. Punta Toro virus, a member of the Phlebovirus genus, induces apoptosis in hepatocytes in vivo and in cultured mammalian cells in vitro [9]. The NSs protein of Punta Toro virus has been shown to induce hepatocyte apoptosis by triggering the extrinsic and intrinsic pathways [10]. Apart from this, NSs proteins in the Bunyaviridae family have been shown to antagonize interferon ␣/␤ production, shut down host cell protein synthesis, and induce the degradation of double-stranded RNA-dependent protein kinase [11,12,13,14,15]. It has been reported that CCHFV induces caspase-3dependent apoptosis [22] and modulates both intrinsic as well as extrinsic pathways of apoptosis in hepatocyte cells [23]. Disruption of the mitochondrial membrane potential by the CCHFV NSs protein is dependent on these key residues

Methods

Results

Conclusion