Mitochondrial targeting of the p13II protein coded by the x-II ORF of human T-cell leukemia/lymphotropic virus type I (HTLV-I)

Abstract

The X region of the HTLV-I genome contains four major open reading frames (ORFs), two of which, termed x-I and x-II, are of still unde®ned biological signi®cance. By indirect immuno¯uorescence and dual labeling with marker proteins, we demonstrate that p13 II , an 87-amino acid protein coded by the x-II ORF, is selectively targeted to mitochondria. Mutational analysis revealed that mitochondrial targeting of p13 II is directed by an atypical 10-amino acid signal sequence that is not cleaved upon import and is able to target the Green Fluorescent Protein to mitochondria. Expression of p13 II results in speci®c alterations of mitochondrial morphology and distribution from a typical string-like, dispersed network to round-shaped clusters, suggesting that p13 II might interfere with processes relying on an intact mitochondrial architecture. Functional studies of mitochondria with the cationic¯uorochrome tetramethylrhodamine revealed that a subpopulation of the cells with p13 II -positive mitochondria show a disruption in the mitochondrial inner membrane potential (Dc), an early event observed in cells committed to apoptosis. Taken together, these results suggest novel virus-cell interactions that might be important in HTLV-I replication and/or pathogenicity.

The human T-cell leukemia virus type 1 p13II protein: effects on mitochondrial function and cell growth

“…18,19 p13 II corresponds to the carboxy-terminal 87 amino acids of p30 II and is produced from a singly spliced mRNA that lacks the Tax initiator codon. 20,21 p13 II also lacks the NLS sequence of p30 II , and is predominantly mitochondrial, 22 although it can occasionally be detected in the nucleus, 14 especially when expressed at high levels (our unpublished observations). Mitochondrial accumulation of p13 II has been documented in a variety of cell types, including T cells, the natural target of HTLV-1 infection (Figure 2).…”

“…Although p13 II 's MTS lies close to its N-terminus and is positively charged, it does not appear to be cleaved. 22 In addition, the observations made for the arginine substitution mutants described above indicate that the p13 II MTS does not require the presence of these positively charged residues and works independently of its ability to fold into an a-helix. 31 These properties do not HTLV-1 p13 II and mitochondria DM D'Agostino et al appear to fit well with any of the inner membrane protein import pathways described above and suggest that the p13 II MTS has peculiar sequence-structure requirements that might direct mitochondrial import through an alternative mechanism.…”

“…22 While a deletion mutant lacking the first 18 residues exhibited mitochondrial targeting, a mutant lacking the first 31 amino acids showed a diffuse pattern throughout the cell, indicating that p13 II 's MTS lies between residues 19 and 31. A tag consisting of residues 21-30 was sufficient to relocalize GFP to mitochondria, thus identifying it as an MTS.…”

“…A tag consisting of residues 21-30 was sufficient to relocalize GFP to mitochondria, thus identifying it as an MTS. 22 Amino-terminal positioning of the MTS appears to be important for it to function efficiently, given that neither p30 II nor an N-terminal truncation mutant of p30 II that lacks the NLS and initiates 56 residues before p13 II 's ATG is able to accumulate in mitochondria. 15 p13 II 's MTS sequence (LRVWRLCTRR) includes four arginines that are predicted to form a positively charged face within an a-helix, thereby imparting amphipathic properties to this region.…”

“…15 p13 II 's MTS sequence (LRVWRLCTRR) includes four arginines that are predicted to form a positively charged face within an a-helix, thereby imparting amphipathic properties to this region. 22 Circular dichroism spectroscopy of a synthetic peptide spanning residues 9-41 (i.e. including the MTS) revealed that it folds into an a-helix upon exposure to membrane-mimetic solutions containing phospholipids or sodium dodecyl sulfate (SDS) micelles.…”