AEGiS-11IAC: 2-5A synthetase: activation by HIV-1 TAR RNA and RRE RNA and modulation by propentofylline in HIV-1 infected CEM cells.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

2-5A synthetase: activation by HIV-1 TAR RNA and RRE RNA and modulation by propentofylline in HIV-1 infected CEM cells.

Int Conf AIDS 1996 Jul 7-12; 11:6 (abstract no. Mo.A.144)
Leuck J, Scheffer U, Muller WE, Schroder HC; Inst. of Physiological Chemistry, University, Mainz, Germany. Fax: 49-6131-395243. E-mail: leuck@mzdmza.zdv.uni-mainz.de.

OBJECTIVE: To determine the structural elements of HIV-1 RNAs (TAR RNA and RRE RNA) that are able to activate the antiviral 2',5'-oligoadenylate (2-5A) system and to search for compounds that prevent the decrease in interferon (IFN)-inducible 2-5A synthetase activity during later stages of infection.

METHODS: Human T lymphoblastoid CEM cells were infected with HIV-1 (strain HTLVIIIB) and cultivated in the absence or presence of compound and IFN. The number of viable cells was monitored using the MTT colorimetric assay. 2-5A synthetase activity was determined in cell extracts after binding to immobilized poly(I)-poly(C). Formation of RNA-protein complexes was detected in RNase protection and Northwestern assays using in vitro synthesized RRE RNA, TAR RNA and TAR RNA mutants.

RESULTS: The TAR RNA sequence is among those RNAs which bind to and activate 2-5A synthetase. Experiments with TAR mutants revealed that binding of 2-5A synthetase to TAR RNA depends on the length but not the sequence of the double-stranded TAR RNA stem. Binding of 2-5A synthetase isoenzymes (40-46 kDa, 67 kDa, and 100 kDa) to TAR RNA could also be demonstrated in RNase protection and Northwestern assays with cytosolic proteins from IFN-treated cells (CEM, HeLa, and other cell lines). The effect of TAR on 2-5A synthetase was abolished in the presence of Tat protein. The 2-5A synthetase was also found to be activated by the RRE sequence of HIV-1 env RNA. Addition of recombinant Rev protein suppressed complex formation between RRE RNA and 2-5A synthetase. Previously we found that the activity of the 2-5A forming 2-5A synthetase and the levels of 2-5A transiently increase after infection of cells with HIV-1, followed by a strong decrease simultaneous with the onset of virus production. In CEM cells the decrease in IFN-inducible 2-5A synthetase activity and protein could be retarded in the presence of the cAMP phosphodiesterase inhibitor propentofylline.

CONCLUSIONS: Both RRE RNA and TAR RNA were able to activate IFN-inducible 2-5A synthetase. Our results indicate that cAMP is involved in control of 2-5A metabolism. The decrease in 2-5A synthetase activity during later stages of infection could be retarded by xanthine derivatives.

Keywords: AEGIS, HIV-1, Gene Products, tat, RNA, Double-Stranded, Xanthines, Oligoribonucleotides, Endoribonucleases, Ligases, Interferons, propentofylline, 2',5'-oligoadenylate, Human, In Vitro, ICA11

960707
MoA144