Differential expression of alternatively spliced pX mRNAs in HTLV-I-infected cell lines

Abstract

Human T cell leukemia/lymphotropic virus (HTLV) is a complexstudies have demonstrated the presence of p30, p13 and p12 9 kb human retrovirus with at least eight alternatively spliced in addition to the previously recognized Tax, Rex and p21 rex mRNAs expressed from the 3Ј or pX region of the genome.proteins. 3,5,7 A unique splice site has also been detected 17 Cell linesHAM) 2 is a complex 9 kb human retrovirus. The degree of complexity of the viral genome has recently been recognized Several previously described IL-2-dependent and IL-2-indewith the detection by several groups of at least eight alternapendent HTLV-I-infected T cell lines were utilized in these tively spliced mRNAs expressed from the 3Ј or pX region of studies. They included the IL-2-independent lines C10/MJ the genome. 3-6 These mRNAs allow for the expression of established from a patient with ATL (who subsequently novel proteins from the previously recognized pX open readdeveloped TSP/HAM), 11 NS1 established from a healthy seroing frames I and II in addition to Tax, Rex and p21 rex encoded positive individual, 12 C8166-45 established from a patient from orf III and IV. The alternatively spliced messages include:with ATL and containing defective proviruses 13 and LAF, the doubly spliced pX-tax/rex with the coding capacity for established from a patient with TSP/HAM. 5 The IL-2-depenTax, Rex and p21 rex ; the singly spliced pX-orf I encoding a dent cell lines, N1185 and N1186, were established by coprotein, p12, and the doubly spliced pX-rex-orf I coding for cultivation of PBMCs from two patients with ATL with cord p12 and with the potential for encoding a 152 amino acid blood cells. 4 The cells were maintained in RPMI with 10% protein (Rex-orf I or Rof), the singly spliced pX-orf II encoding FCS with the addition of Il-2 (20 units/ml, Boehringer p13; the doubly spliced pX-tax-orf II encoding a larger protein Mannheim, Indianapolis, IN, USA) for the IL-2-dependent (p30, Tax-orf II or Tof) which utilizes the initiation codon of cell lines. Tax; and the singly spliced pX-p21 rex , a monocistronic message encoding p21 rex of unknown function. In vitro transfection RNA preparation

Limiting amounts of p27Kip1 correlates with constitutive activation of cyclin E-CDK2 complex in HTLV-I-transformed T-cells

“…We, as well as others, have previously shown that p21 Waf1 expression is increased in HTLV-Iinfected T-cells and that Tax can induce its expression in a p53-independent manner (Akagi et al, 1996;Cereseto et al, 1996). Increasing amounts of p21 Waf1 correlated with constitutive activation of the cyclin E complex, suggesting that, at a particular stoichiometry, p21 Waf1 may lower the activation threshold of the cyclin E-CDK2 complex (La Baer et al, 1997;Macleod et al, 1995;Zhang et al, 1994).…”

“…In contrast, p21 Waf1 was consistently found highly expressed in HTLV-I-infected T-cells regardless of their requirement for IL-2 and presumably the viral transactivator Tax contributes to maintain high level of p21 mRNA (Akagi et al, 1996;Cereseto et al, 1996).…”

“…Although at this stage it is not possible to rule out the possibility that viral genes may be responsible for low protein levels of p27 Kip1 , it is unlikely that, as in the case of p21 Waf1 (Cereseto et al, 1996), Tax, the viral transcriptional transactivator, could be involved. p27 Kip1 mRNA levels do not di er in HTLV-I-infected dependent or independent cells, and modulation of the expression of other HTLV-I accessory genes does not correlate with the IL-2-dependent/independent status of the HTLV-I-infected T-cells .…”

“…Recombinant human IL-2 (20 U/ml) was added to the growth medium of N1186 (Migone et al, 1995) cells and to PBMCs. For the starvation experiments, PBMCs, N1186, and N1186-94 (Migone et al, 1995) and N1185, LAF, C10/MJ, MJ, C91/ PL, MT-2, and C8166 (Cereseto et al, 1996) were grown for three days in media containing 0.1% of serum with no IL-2. When indicated, rapamycin (60 nM) (Calbiochem, La Jolla, CA, USA) or LY294002 (40 mM for 24 h) (Calbiochem) dissolved in DMSO was added to the culture media and equal volumes of DMSO were added to mock cultures.…”

“…Cell extracts were prepared as described (Cereseto et al, 1996) and protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). For immunoprecipitations, 900 mg of protein lysate was incubated with speci®c antibodies; the immunocomplexes were collected using 20 ml of protein G-Sepharose (Boehringer-Mannheim) and washed three times in lysis bu er.…”