vIRF-1 localization to mitochondria.

(A) Homogenates of HEK293T cells transfected with wild-type or BBD-mutated (GK₁₇₉AA) vIRF-1 (or empty vector control, vIRF-1-negative) were subjected to differential and Optiprep gradient centrifugation to isolate enriched mitochondrial fractions (see Materials and Methods). These were analyzed by immunoblotting with vIRF-1-specific rabbit antiserum for the presence of vIRF-1 protein, and also with antibody to mitochondrial protein VDAC1 [voltage-dependent anion-selective channel protein 1; integral mitochondrial outer membrane (int. OM) protein] to provide a positive control. Normalization of vIRF-1 amounts in lysates versus mitochondrial fractions from these transfected cells was achieved using a ratio of 1∶50 (bottom). (B) In vitro mitochondrial binding assays using enriched mitochondria, untreated or pre-treated with proteinase K (Prot. K), and recombinant T7-tagged vIRF-1 for assessment of the requirement for surface protein integrity for mitochondrial binding by vIRF-1. Total protein released from mitochondria was precipitated with trichloroacetic acid (TCA) for direct quantitative comparison with protein from mitochondrial pellets. Blots were probed with antibodies to mitochondrial outer membrane (OM)-associated Bax and inner membrane (IM)-localized prohibitin to provide controls for appropriate fractionation, mitochondrial integrity, and proteinase K activity. (C) Generation and Western analysis of mitochondrial preparations from HHV-8⁺ BCBL-1-TRE/RTA cells [70] revealed mitochondrial association of endogenous vIRF-1, both in resting (latent) and lytically reactivated (+Dox) cultures; the latter, as expected, expressed higher levels of vIRF-1. In both cases, mitochondrial-associated vIRF-1 was susceptible to protease digestion, consistent with peripheral binding to mitochondria. Immunodetection of Bim verified peripheral protein susceptibility to proteinase K digestion. (D) Mitochondrial localization of vIRF-1 as determined using immunoflourescence assay for detection of vIRF-1 and mitochondrial marker TOM20 in TIME-TRE/vIRF-1 endothelial cells, +Dox. Arrows indicate examples of vIRF-1/TOM20 co-localization. (E) Western analyses of total cell and mitochondrial extracts of HHV-8⁺ TIME-TRE/RTA and BCBL1-TRE/RTA cells, untreated or treated with Dox for 2 days, were undertaken to quantify the relative amounts of mitochondrial-localized vIRF-1 in these latently and lytically infected cells. For TIME and BCBL-1 cells, 10- and 50-fold excess of mitochondrial extract over total extract, respectively, was loaded onto the gels to achieve near normalization. Relative signal intensities of bands were obtained from digitally captured images and calculated values of mitochondrial relative to total vIRF-1 levels are shown under the vIRF-1 blots. vIRF-1 was detected in latency (−Dox) only in the BCBL-1 (PEL) cells, and mitochondrial∶total vIRF-1 was increased upon Dox addition. Immunoblotting for VDAC1 (mitochondrial), histone deacetylase-1 (HDAC1, nuclear), calreticulin (endoplasmic reticulum) and β-actin (cytoplasmic) provided quality controls for mitochondrial and total cell extracts. (Arrowhead, vIRF-1; *non-specific). (F) A similar experiment in HHV-8⁺ TIME-TRE/RTA cells, demonstrating mitochondrial localization of Bid, in addition to vIRF-1, in lytically reactivated cultures.