Antisense expression cloning of HIV host factor genes.
Int Conf AIDS 1996 Jul 7-12; 11:57 (abstract no. Mo.A.1010) Schalk HJ, Scheinert P, Thiesen J, Bendrat E; Bernhard-Nocht-Institute for Tropical Medicine Virology, Hamburg, Germany. Fax: 49-70-31182309.
Besides its receptor CD4 HIV presumably needs additional membrane bound and intracellular receptors. To isolate genes for essentially needed host factors, we developed a three step cell genetic screen based on the expression of antisense RNA libraries. 1.) We constructed very small cDNA antisense libraries of cellular mRNAs by normalisation and improved subtraction techniques. 2.) We elaborated an episomal minivector system that permits electroporation of complete antisense libraries (greater than 106) into cells. 3.) We constructed a HIV suicide cell line to select antisense vectors with anti-HIV activity from large libraries. From electroporated cells vectors are reisolated, that rendered cells resistant against infection by expression of an antisense-RNA essential for HIV but dispensable for the cell. The efficiency of the antisense expression and selection system was confirmed using antisense vectors directed against the known host factor CD4. Due to their genetic stability cellular host factor genes represent promising therapeutic targets. Besides its receptor CD4 HIV presumably needs additional membrane bound and intracellular receptors. To isolate genes for essentially needed host factors, we developed a three step cell genetic screen based on the expression of antisense RNA libraries. 1.) We constructed very small cDNA antisense libraries of cellular mRNAs by normalisation and improved subtraction techniques. 2.) We elaborated an episomal minivector system that permits electroporation of complete antisense libraries (greater than 106) into cells. 3.) We constructed a HIV suicide cell line to select antisense vectors with anti-HIV activity from large libraries. From electroporated cells vectors are reisolated, that rendered cells resistant against infection by expression of an antisense-RNA essential for HIV but dispensable for the cell. The efficiency of the antisense expression and selection system was confirmed using antisense vectors directed against the known host factor CD4. Due to their genetic stability cellular host factor genes represent promising therapeutic targets.
Keywords: AEGIS, HIV, RNA, Antisense, Carrier Proteins, HIV-1, Antigens, CD4, host factors, Cell Line, HIV Long Terminal Repeat, HIV Core Protein p24, RNA, Messenger, Anti-HIV Agents, genetics, ICA11
