Isolation, Cloning, and Complete Nucleotide Sequence of a Phenotypically Distinct Brazilian Isolate of Human T-Lymphotropic Virus Type II (HTLV-II)

Abstract

Analysis of human T-lymphotropic virus type II (HTLV-II) isolates from North America and Europe have demonstrated the existence of two molecular subtypes of the virus, HTLV-IIa and HTLV-IIb. Recently, studies on HTLV-II infections in Brazil have revealed isolates that are related phylogenetically to the HTLV-IIa subtype but have a HTLV-IIb phenotype with respect to the transactivating protein, tax. To more clearly define this relationship, HTLV-II was isolated from peripheral blood of an IVDA from Sao Paulo, Brazil (SP-WV), and the complete provirus was cloned and sequenced. Comparison of HTLV-II(SP-WV) nucleotide sequences to other available complete HTLV-II proviral sequences revealed that HTLV-II(SP-WV) is most closely related to HTLV-II(Mo), the prototypic HTLV-IIa subtype sequence. Phylogenetic analysis of LTR, env, and tax regions unequivocally demonstrated that HTLV-II(SP-WV) and all other Brazilian sequences examined are members of the IIa subtype. The predicted amino acid sequences of the major coding regions of HTLV-II(SP-WV) are also most closely related to HTLV-II(Mo), with the important exception of tax. The tax protein encoded by HTLV-II(SP-WV) is 96-99% identical to the tax of IIb isolates and is similar in that it has an additional 25 amino acids at the carboxy-terminus compared to the HTLV-II(Mo) tax with which it shares 91% identity. Analysis of tax stop codon usage of a number of HTLV-IIa isolates from North American, Europe, and Brazil demonstrated that isolates from the last region appear to be unique in their extended tax phenotype. It could be demonstrated that the extended tax proteins in the HTLV-IIb and Brazilian isolates had equivalent ability to transactivate the viral LTR, and studies with deletion mutants indicated that the extended C-terminus is not essential for transactivation. In contrast, the HTLV-IIa tax was found to have a greatly diminished ability to transactivate the viral LTR, which appeared to be a consequence of reduced expression of the protein. The studies show that although the Brazilian strains do not represent an entirely new subtype based on nucleotide sequence analysis they are a phenotypically unique molecular variant within the HTLV-IIa subtype.

Aberrant Activation of the Interleukin-2 Autocrine Loop through the Nuclear Factor of Activated T Cells by Nonleukemogenic Human T-Cell Leukemia Virus Type 2 but Not by Leukemogenic Type 1 Virus

“…The proteins in the gel were electrotransferred onto a polyvinylidene difluoride membrane. The membrane was incubated with 5% skim milk for 1 h at room temperature to inhibit nonspecific binding and was further incubated with either anti-Tax1 mouse monoclonal antibody (Taxy-7) (37) or rabbit anti-Tax2B polyclonal serum (22). After being washed with TBS-T (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% Tween 20), the membranes were incubated with either anti-mouse (for Tax1) or anti-rabbit (for Tax2B) immunoglobulin G conjugated with horseradish peroxidase (Bio-Rad Technologies).…”

Human T-Cell Leukemia Virus Type 2 (HTLV-2) Tax Protein Transforms a Rat Fibroblast Cell Line but Less Efficiently than HTLV-1 Tax

“…The primers used to amplify the tax2A gene were ttgaattcagatctCCATGGCCCATTTCCCAGGATTCGGA and tggatc cTTTTAGGCCGATGACTCGT. The tax2B cDNA was derived from plasmid pCAGGS-Tax2B (29). Lowercase letters in the sequence are the restriction enzyme sites for EcoRI and BamHI to facilitate the subcloning of the fragments into the expression plasmid and do not exist in the pC-Xc plasmid sequence.…”

“…Cell lysates (25 g) prepared from Rat-1 cells were size separated by electrophoresis under reducing conditions in 10% polyacrylamide gel with sodium dodecyl sulfate, and the proteins in the gel were electronically transferred onto a polyvinylidene difluoride membrane. The membrane was incubated with Block Ace (Dainippon Seiyaku, Suita, Osaka, Japan) for 1 h at room temperature to inhibit nonspecific binding and was further incubated with either anti-Tax mouse monoclonal antibody (Taxy-8) (55) or rabbit anti-Tax2B polyclonal serum (29). After being washed with TBS-T (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.05% Tween 20), the membranes were further incubated with either anti-mouse or anti-rabbit immunoglobulins conjugated with horseradish peroxidase (Bio-Rad Technologies, Richmond, Calif.).…”

“…HTLV-2 also encodes a trans-activator protein, Tax2 (29,48), which exhibits more than 70% amino acid identity with Tax1. Ross et al (41) showed that mutation of the tax2 gene in an infectious HTLV-2 molecular clone eliminates the transforming activity of Tax2 in primary human T cells.…”

High Frequencies of Functionally Competent Circulating Tax-Specific CD8+ T Cells in Human T Lymphotropic Virus Type 2 Infection

“…For quantitation of HTLV-2 Tax gene, the primers Tax2C-S (5Ј-CGATTGTGTACAGGCCGATTG-3Ј, positions 7275-7295), Tax2C-AS (5Ј-CAGGAGGGCATGTCGATGTAG-3Ј, positions 7350 -7330), and the probe Tax2CP (5Ј-FAM-TGTCCCGTCTCAGGTGGTCTATGTTC CA-BHQ-3Ј, positions 7297-7324) were designed using the software Primer Express 3.0 (Applied Biosystems), based on a previously published sequence from a Brazilian HTLV-2c isolate (GenBank accession no. AF139382) (21). Quantification of a human albumin gene fragment was used as a reference gene.…”