Mitochondrial lipid raft-like microdomains, experimentally also termed mitochondrial detergent-resistant membrane fractions (mDRM), play a role as platforms for recruiting signaling molecules involved in antiviral responses such as apoptosis and innate immunity. Viruses can modulate mitochondrial functions for their own survival and replication. However, viral regulation of the antiviral responses via mDRM remains incompletely understood. Here, we report that human herpesvirus 8 (HHV-8) gene product viral interferon regulatory factor 1 (vIRF-1) is targeted to mDRM during virus replication and negatively regulates the mitochondrial antiviral signaling protein (MAVS)-mediated antiviral responses. The N-terminal region of vIRF-1 interacts directly with membrane lipids, including cardiolipin. In addition, a GxRP motif within the N terminus of vIRF-1, conserved in the mDRM-targeting region of mitochondrial proteins, including PTEN-induced putative kinase 1 (PINK1) and MAVS, was found to be important for vIRF-1 association with mitochondria. Furthermore, MAVS, which has the potential to promote vIRF-1 targeting to mDRM possibly by inducing cardiolipin exposure on the outer membrane of mitochondria, interacts with vIRF-1, which, in turn, inhibits MAVS-mediated antiviral signaling. Consistent with these results, vIRF-1 targeting to mDRM contributes to promotion of HHV-8 productive replication and inhibition of associated apoptosis. Combined, our results suggest novel molecular mechanisms for negative-feedback regulation of MAVS by vIRF-1 during virus replication. H uman herpesvirus 8 (HHV-8), also called Kaposi's sarcomaassociated herpesvirus (KSHV), is a pathogenic DNA virus associated with Kaposi's sarcoma (KS) and the B cell malignancies primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD), which often occur in immunocompromised individuals, such as those with human immunodeficiency virus type 1 (HIV-1) infection or undergoing organ transplantation (1, 2). Virus productive replication, in addition to latency, is important for maintaining viral load within the host and also for HHV-8-associated pathogenesis. Successful virus replication is in large part achieved by the ability of viruses to counteract antiviral responses of the host cells, such as apoptosis and innate immune responses. HHV-8 encodes a number of proteins expressed during the lytic cycle that have demonstrated or potential abilities to promote virus productive replication via inhibition of apoptosis and innate immune signaling pathways (3). Among them, viral interferon (IFN) regulatory factor 1 (vIRF-1) is believed to play crucial roles in blocking interferon and other stress responses to virus infection and replication by negatively interacting with cellular stress signaling proteins, including p53, ATM, IRF-1, IRF-3, GRIM19, SMAD3,. In addition, we discovered that vIRF-1 localizes to the outer mitochondrial membrane (OMM) and inhibits the mitochondrial intrinsic apoptosis pathway via its inhibitory interaction with proapo...