A lysine-to-arginine change found in natural alleles of the human T-cell lymphotropic/leukemia virus type 1 p12(I) protein greatly influences its stability

Abstract

The HTLV-1 singly spliced open reading frame I protein, p12(I), is highly unstable and appears to be necessary for persistent infection in rabbits. Here we demonstrate that p12(I) forms dimers through two putative leucine zipper domains and that its stability is augmented by specific proteasome inhibitors. p12(I) is ubiquitylated, and mutations of its unique carboxy-terminus lysine residue to an arginine greatly enhance its stability. Interestingly, analysis of 53 independent HTLV-1 strains revealed that the natural p12(I) alleles found in ex vivo samples of tropical spastic paraparesis-HTLV-1-associated myelopathy patients contain a Lys at position 88 in some cases, whereas arginine is consistently found at position 88 in HTLV-1 strains from all adult T-cell leukemia-lymphoma (ATLL) cases and healthy carriers studied. This apparent segregation of different alleles in tropical spastic paraparesis-HTLV-associated myelopathy and ATLL or healthy carriers may be relevant in vivo, since p12(I) binds the interleukin-2 receptor beta and gammac chains, raising the possibility that the two natural alleles might affect differently the regulation of these molecules.

FIG. 1

Amino-acid sequence of the singly spliced ORFI putative p12^I protein. The amino acid single code is used. TM-1 and TM-2 stand for the putative TM regions of the protein. The two amino acids (FL) are boxed because they correspond to possible destabilizing residues according to the N degron rule.

FIG. 2

High-molecular-weight proteins detected in immunoprecipitation of wild-type and p12^I mutants. Radio immunoprecipitation was performed by using anti-AU1 Ab in cells transfected with p12^I_AU1 (lane 1), vector only (lane 4), and the Δ14 and Δ35 mutants (lanes 2 and 3, respectively). The thick and thin arrows indicate the positions of extra bands in the p12^I wild-type transfectants (lane 1) and p12^I mutant transfectants (lanes 2 and 3).

FIG. 3

p12^I regions involved in intermolecular interaction of p12^I. The top part of the figure is a graphical representation of p12^I deletion mutants. The numbers refer to the amino acid position within p12^I. The black bars encompass the putative TM domains of p12^I. The gray areas stand for the epitope location. IP, immunoprecipitation; WB, Western blot.

FIG. 4

p12^I regions involved in intermolecular interaction of p12^I (continued). See the legend to Fig. 3 for an explanation of panels, symbols, and abbreviations.

FIG. 5

p12^I is ubiquitylated and stabilized by proteasome inhibitors. 293T cells were transiently transfected with p12^I HA1 expression plasmid or pME vector control and lysed in 1× RIPA buffer 24 h later. (A) An antiubiquitin immunoprecipitate of cell lysates is shown in the top panel. The bottom panel shows results with 50 μg of total cell lysate. An anti-HA1 Western blot was performed for both membranes. (B and C) An anti-HA1 immunoprecipitate of cell lysates was performed, and the immunoprecipitates were split into duplicate membranes. (B) Antiubiquitin Western blot. (C) Blot probed with α-HA1 Ab. Arrows indicate superimposable bands present on both blots. + and −, presence or absence, respectively, of drugs.

FIG. 6

Sensitivity of p12^I mutants and p12^IK88 or p12^IR88 to proteasome inhibitors. Panels A, B, and C show the results of Western blot assays with α-HA1 or α-AU1 Ab in transiently transfected 293T cells. In panel C, a Western blot was preceded by α-HA1 immunoprecipitation. p12^IR88 is a mutant that encodes an R instead of a K in codon 88. (A) p12^I mutants Δ47 and Δ35 are stabilized by proteasome inhibitors, while +70 and 36–70 mutants lacking the Lys 88 residue are not. (B) The steady-state level of the isogene mutant p12^IR88 is greatly increased compared to p12^IK88. (C) High-molecular-weight forms of p12^I are clearly present in cells transfected with p12^IK88 but are absent in those expressing p12^IR88. (D) The steady-state levels of p12^IK88 and p12^IR88 in the presence of all three proteasome inhibitors at the concentration indicated in the Materials and Methods are shown.

FIG. 7

Differential stability of p12^IK88 and p12^IR88. Western blot analysis with anti-HA1 Ab of total proteins from transfected cells after treatment with 10 μg of cycloheximide per ml for the indicated time intervals. The single substitution of an Arg for a Lys at position 88 significantly increased the half-life of the protein. The experiment was repeated three to four times for each protein.

FIG. 8

Natural allelic mutation at position 88 in samples obtained from HTLV-1-infected patients. Southern blot analysis of PCR product from the HTLV-I ORFI before and after ApaI cleavage. ATL-1* and ATL-2* indicate patients with both TSP-HAM and ATLL.