Human Herpesvirus 8 Interferon Regulatory Factor-Mediated BH3-Only Protein Inhibition via Bid BH3-B Mimicry

Abstract

Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of host cells. For HHV-8, viral interferon regulatory factor-1 (vIRF-1) contributes to this process in part via inhibitory interactions with BH3-only protein (BOP) Bim, recently identified as an interaction partner of vIRF-1. Here we recognize that the Bim-binding domain (BBD) of vIRF-1 resembles a region (BH3-B) of Bid, another BOP, which interacts intramolecularly with the functional BH3 domain of Bid to inhibit it pro-apoptotic activity. Indeed, vIRF-1 was found to target Bid in addition to Bim and to interact, via its BBD region, with the BH3 domain of each. In functional assays, BBD could substitute for BH3-B in the context of Bid, to suppress Bid-induced apoptosis in a BH3-binding-dependent manner, and vIRF-1 was able to protect transfected cells from apoptosis induced by Bid. While vIRF-1 can mediate nuclear sequestration of Bim, this was not the case for Bid, and inhibition of Bid and Bim by vIRF-1 could occur independently of nuclear localization of the viral protein. Consistent with this finding, direct BBD-dependent inactivation by vIRF-1 of Bid-induced mitochondrial permeabilization was demonstrable in vitro and isolated BBD sequences were also active in this assay. In addition to Bim and Bid BH3 domains, BH3s of BOPs Bik, Bmf, Hrk, and Noxa also were found to bind BBD, while those of both pro- and anti-apoptotic multi-BH domain Bcl-2 proteins were not. Finally, the significance of Bid to virus replication was demonstrated via Bid-depletion in HHV-8 infected cells, which enhanced virus production. Together, our data demonstrate and characterize BH3 targeting and associated inhibition of BOP pro-apoptotic activity by vIRF-1 via Bid BH3-B mimicry, identifying a novel mechanism of viral evasion from host cell defenses.

Genome-Wide Mapping of the Binding Sites and Structural Analysis of Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 2 Reveal that It Is a DNA-Binding Transcription Factor

“…Moreover, KS is the first tumor to be associated with HIV infection and the most common AIDS-associated malignancy in sub-Saharan Africa and the second most common cancer in HIV-infected patients (2)(3)(4)(5). KSHV has developed a unique mechanism to subvert host antiviral immune responses by encoding four homologues (viral interferon regulatory factors [vIRF1 to -4]) of cellular interferon regulatory factors (c-IRFs) with a cluster of KSHV open reading frames (ORFs), K9, K11/11.1, K10.5/10.6, and K10, respectively (6)(7)(8). vIRFs have been demonstrated to be effective inhibitors of interferon signaling and regulators of cellular oncogenic pathways, which may contribute to KSHV infection, as well as pathogenesis (4,9,10).…”

“…However, some evidence has suggested that the short-form vIRF2 (K11.1) was capable of directly binding to a consensus NF-B binding site, but not to an ISRE (8). vIRF1 has been shown to bind some DNA oligonucleotides in vitro and can interact with the K3-viral dihydrofolate reductase-viral interleukin 6 promoter region in the KSHV genome (20).…”

Distinct Roles of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interferon Regulatory Factors in Inflammatory Response and Cancer

“…In contrast to Bim, vIRF1 was unable to mediate nuclear sequestration of Bid protein. Direct functional inhibition of Bid by vIRF1 was demonstrated by the ability of vIRF1 to block Bid-induced mitochondrial permeabilization in vitro (69). Furthermore, Western blot analysis of mitochondrial preparations from KSHV-positive BCBL-1 cells, or TIME cells, revealed mitochondrial association of endogenous vIRF1 in latent and lytic cultures.…”

“…Thus, vIRF1-induced nuclear localization and inactivation of Bim represents a novel mechanism of viral evasion from antiviral defenses of the host. Recently, the Bim-binding region (BBD) of vIRF1 was shown to interact also with the BH-3 domain of other BOPs, e.g., Bid, Bik, Bmf, Hrk, and Noxa (69). In contrast to Bim, vIRF1 was unable to mediate nuclear sequestration of Bid protein.…”

“…Furthermore, Western blot analysis of mitochondrial preparations from KSHV-positive BCBL-1 cells, or TIME cells, revealed mitochondrial association of endogenous vIRF1 in latent and lytic cultures. Thus, Bid and Bim are contributors to negative regulation of KSHV infection, and their targeting by vIRF1 may be important for productive virus replication (69). vIRF1 and GRIM19.…”