Mitochondrial localization of p13^II. (a) Primary rat embryo fibroblasts transfected with the p13^II expression plasmid pSGp13^II using Fugene 6 (Roche) are shown; (b) A human T-cell (Jurkat TetOn cell line; Clontech) transfected with the p13^II expression plasmid pcDNAp13^II using MegaFectin (Q.Biogene) are shown. After formaldehyde-fixation and permeabilization with Nonidet P40, the cells were analyzed by indirect immunofluorescence using a rabbit antibody recognizing p13^II and Alexa 488-conjugated anti-rabbit secondary antibody (Molecular Probes); to visualize mitochondria, the cells were counterstained with mouse anticytochrome c antibody (Pharmingen; a) or goat anti-Hsp60 antibody (Santa Cruz; b) and appropriate Alexa 546 red-conjugated secondary antibodies (Molecular Probes). In addition to verifying colocalization of 13^II with the mitochondrial marker proteins, (a) shows the dramatic changes in mitochondrial morphology induced by the protein

Possible effects of p13^II on apoptosis and Ras function. The Fas and Fas-ligand (FasL) interaction results in activation of sphingomyelinase and generation of ceramide, which leads to PTP sensitization and is a known activator of Ras. Ras function is dependent on its post-translational farnesylation, mediated by farnesyl transferase (FTase). The farnesyl substrate group (farnesyl pyrophosphate) is synthesized through the mevalonate–squalene pathway by farnesyl pyrophosphate synthetase (FPPS), a cellular binding partner of p13^II. p13^II might affect these pathways by enhancing PTP sensitization and/or by controlling Ras farnesylation/function through its binding to FPPS. FTI, farnesyl transferase inhibitor

Expression of the x-II ORF of HTLV-1. (a) HTLV-1 genome, ORFs, transcripts, and protein products are shown. The exon boundaries are indicated below each transcript, with nucleotide numbering starting at the first nucleotide in the R region of the LTR. (b) Protein sequences of p30^II (expressed from mRNA 1-2-B; in grey letters) and p13^II (expressed from mRNA 1-C; in black letters) coded by HTLV-1 molecular clone ATK are shown. The p13^II sequence spans nucleotides 6936–7197 and corresponds to the C-terminal 87 amino acids of p30^II. The bipartite NLS of p30^II and the MTS of p13^II are underlined. (c) Differences in the p13^II amino-acid sequence of the indicated viruses compared to ATK p13^II (see b) are shown. ATK and ATL-YS are proviruses cloned directly from peripheral blood lymphocytes of Japanese ATLL patients.3,4 CS-HTLV is a full-length, infectious provirus derived from a North American ATLL patient.5 HS-35 is a molecular clone obtained from an infected patient of Caribbean origin.6 IMEL5 is an HTLV-1 isolate from a healthy Melanesian Solomon Islander.7 Isolates HH, TE, TG and TD are from healthy carriers (HH) and HAM/TSP patients;8,9 changes are based on published analyses of the portion of exon 3 (the second Tax/Rex exon) overlapping with exon C

Working model of p13^II’s mechanism of action at the mitochondrial level. Insertion of p13^II into the inner mitochondrial membrane either directly or indirectly induces a rapid potential-dependent K⁺ current in mitochondria. This influx of positive charges may lead to mitochondrial depolarization which, in turn, may (i) increase sensitivity to apoptosis by lowering the threshold for PTP opening; and (ii) alter the Ca²⁺ uptake capacity of mitochondria, thus changing intracellular Ca²⁺ homeostasis and signaling. PM, plasma membrane; ER, endoplasmic reticulum; Δψ, inner mitochondrial membrane potential; PTP, permeability transition pore; grey dots indicate Ca²⁺, asterisks indicate proapoptotic factors contained in the mitochondrial intermembrane space, for example cytochrome c, apoptosis-inducing factor, SMAC/Diablo