Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells.
Tat-mediated trans-activation of the HIV-1 long terminal repeat (LTR) occurs through the phosphorylation of the carboxy-terminal domain of the RNA polymerase II. The kinase complex, pTEFb, composed of cyclin T1 (CycT1) and CDK9, mediates this process. The trans-activation response (TAR) RNA-binding protein 2 (TRBP2) increases HIV-1 LTR expression through TAR and protein kinase R (PKR) binding, but not through interactions with the Tat-CycT1-CDK9 complex. TRBP2 and the Tat-CycT1-CDK9 complex have overlapping binding sites on TAR RNA. TRBP2 and CycT1 increased Tat trans-activation in NIH 3T3 cells with additive effects. Upon transfection of HIV-1 pLAI, pNL4-3, pMAL, and pAD molecular clones, reverse transcriptase (RT) activity and p24 concentration were decreased 200- to 900-fold in NIH 3T3 cells compared with HeLa cells in both cells and supernatants. In murine cells, cotransfection of the HIV clones with CycT1 or TRBP2 increased modestly the expression of RT activity in cell extracts. The analysis of Gag expression in murine cells transfected with CycT1 compared with human cells showed a 20-fold decrease in expression and a strong processing defect. The expression of both CycT1 and TRBP2 had a more than additive activity on RT function in cell extracts and on viral particle production in supernatant of murine cells. These results suggest an activity of CycT1 and TRBP2 at different steps in HIV-1 expression and indicate the requirement for another posttranscriptional factor in murine cells for full HIV replication.
Battisti
Pier-Luigi
PL
Molecular Oncology Group, Lady Davis Institute for Medical Research, Montréal, Québec H3T 1E2, Canada.
Daher
Aïcha
A
Bannwarth
Sylvie
S
Voortman
Johannes
J
Peden
Keith W C
KW
Hiscott
John
J
Mouland
Andrew J
AJ
Benarous
Richard
R
Gatignol
Anne
A
D000818:Animals; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9 / Q000502:physiology; D016213:Cyclins / Q000494:pharmacology*; D004357:Drug Synergism; D015683:Gene Products, gag / Q000378:metabolism; D015696:Gene Products, tat / Q000494:pharmacology; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D016601:RNA-Binding Proteins / Q000494:pharmacology*; D015533:Transcriptional Activation; D014779:Virus Replication / Q000187:drug effects; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein
D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D016213:Cyclins; D015683:Gene Products, gag; D015696:Gene Products, tat; D016601:RNA-Binding Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein; C503684:CCNT1 protein, human; C503685:Ccnt1 protein, mouse