The Hepatitis C Virus NS2 Protein Is an Inhibitor of CIDE-B-induced Apoptosis

Abstract

Chronic hepatitis C virus (HCV) infection frequently leads to liver cancer. To determine the viral factor(s)potentially involved in viral persistence, we focused our work on NS2, a viral protein of unknown function. To assign a role for NS2, we searched for cellular proteins that interact with NS2. Performing a two-hybrid screen on a human liver cDNA library, we found that NS2 interacted with the liver-specific pro-apoptotic CIDE-B protein. Binding specificity of NS2 for CIDE-B was confirmed by cell-free assays associated with colocalization studies and coprecipitation experiments on human endogenous CIDE-B. CIDE-B, a member of the novel CIDE family of apoptosis-inducing factors, has been reported to show strong cell death-inducing activity in its C-terminal domain. We show that this CIDE-B killing domain is involved in the NS2 interaction. NS2 binding was sufficient to inhibit CIDE-B-induced apoptosis because an NS2 deletion mutant unable to interact with CIDE-B in vitro lost its capacity to interfere with CIDE-B cell death activity. Although it has been reported that CIDE-Binduced apoptosis is characterized by mitochondrial localization, the precise apoptotic mechanism remained unknown. Here, we show that CIDE-B induced cell death in a caspase-dependent manner through cytochrome c release from mitochondria. Furthermore, we found that NS2 counteracted the cytochrome c release induced by CIDE-B. In vivo, the CIDE-B protein level was extremely low in adenovirus-infected transgenic mice expressing the HCV polyprotein compared with that in wild-type mice. We suggest that NS2 interferes with the CIDE-B-induced death pathway and participates in HCV strategies to subvert host cell defense.

Functional analysis of FSP27 protein regions for lipid droplet localization, caspase-dependent apoptosis, and dimerization with CIDEA

“…In the initial cloning report for CIDEA and CIDEB, it was indicated that apoptosis induced by transient expression of CIDEA in 293T cells was caspase independent due to the failure of caspase inhibitor, when added at 8 h posttransfection, to block CIDEA-mediated apoptosis (11). On the other hand, a study of CIDEB supported a caspase-dependent mechanism of action in that activity of caspase-3, and release of mitochondrial cytochrome c occurred upon transient expression of CIDEB in COS cells (8). It remains to be determined whether the apoptotic mechanism of CIDEA differs from that for FSP27 and CIDEB or whether the early report of caspase independence for CIDEA might be due to the rather late timing of addition of caspase inhibitor in that study (11).…”

“…Studies to date indicate that protein-protein interactions are important to CIDE protein function, and both CIDEA and CIDEB have been studied somewhat in this regard. Homo-and heterodimeric interaction of CIDEA and/or CIDEB (i.e., CIDEA:CIDEA, CIDEB:CIDEB, and CIDEA:CIDEB) has been reported (5,8,11,19,27). CIDEB interacts with viral protein NS2 (8) and apolipoprotein B (42) and CIDEA with AMPK (27); such interactions appear to impact the physiological function of these CIDE partner proteins.…”

“…Homo-and heterodimeric interaction of CIDEA and/or CIDEB (i.e., CIDEA:CIDEA, CIDEB:CIDEB, and CIDEA:CIDEB) has been reported (5,8,11,19,27). CIDEB interacts with viral protein NS2 (8) and apolipoprotein B (42) and CIDEA with AMPK (27); such interactions appear to impact the physiological function of these CIDE partner proteins. In cases where protein regions for CIDEA-and CIDEB-mediated interactions have been mapped, they involve the respective CIDE C region (5,8,11,27,42).…”

“…CIDEB interacts with viral protein NS2 (8) and apolipoprotein B (42) and CIDEA with AMPK (27); such interactions appear to impact the physiological function of these CIDE partner proteins. In cases where protein regions for CIDEA-and CIDEB-mediated interactions have been mapped, they involve the respective CIDE C region (5,8,11,27,42). CIDEA and CIDEB were initially cloned on the basis sequence homology to the CIDE N domain of DFF45, the inhibitory regulatory subunit of the major apoptotic nuclease DFF40.…”

“…Curiously, the first described function of CIDEs was promotion of apoptosis. FSP27, CIDEA, and CIDEB exert robust apoptotic activity upon ectopic expression in mammalian cells (5,8,11,13,17). CIDE proteins have a region of amino acid sequence homology in their NH 2 -terminal halves, termed the CIDE N domain, that is also present in the major proapoptotic nuclease DFF40 and its inhibitory partner protein DFF45 (11).…”