12th International AIDS Conference Geneva, Switzerland - June 28-July 3, 1998

Int Conf AIDS 1998 Jun 28-Jul 3; 12:8 (abstract no. 11127)

Loewer J, Marschall M, Loewer R, Bergmann S
Paul-Ehrlich-Institut, Langen, Germany.

OBJECTIVES: To detect and characterize intracellular mechanisms which are able to inactivate integrated HIV genomes.

METHODS: A human T-cell line CEM-CM3 (CBH) stably transfected with a single copy of an HIV construct was used. Instead of gag/pol genes the construct carries a marker gene (HPRT) for selection in HAT medium. HPRT expression coupled to viral mRNA expression enables survival of the cell clone (CBH+). Cell derivatives in which expression is suppressed (CBH-) are able to grow in 8-azaguanine-medium.

RESULTS: Some 8-azaguanine resistent cell clones regain the ability to produce HPRT in HAT medium (CBHr+), but fourteen percent of the clones (CBHi-) are irreversibly inactivated and can not adapt to HAT medium again. In contrast to CBHr+ cells, viral mRNAs were not detected in CBHi-cells by Northern Blot analysis. Southern blots with methylationsensitive restriction enzyms correlate with the degree of methylation and the type of inactivation. The irreversible inactivation can be obviated by treatment with the demethylating agent 5'-azacytidine, with TNF-alpha and after transfection with a tat expression plasmid. After change of HAT medium, CBHi+ cells survive, produce viral mRNA again and methylation of the viral genome is diminished.

CONCLUSION: Further studies to define in detail the methylation pattern of the reversibly, the irreversibly inactivated and the reactivated integrated HIV genomes by bisulphite based cytosine methylation analysis will show its contribution to regulation of the HIV construct by methylation.

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