HIV REPORTER CELL LINE FOR SCREENING ANTI-SENSE AGENTS

A SECRETABLE ALKALINE PHOSPHATASE (SPAP) REPORTER GENE WILL BE SUBSTITUTED FOR A PORTION OF THE ENV GENE IN AN HIV PROVIRAL DNA CLONE. EXPRESSION OF THE REPORTER GENE WILL BE REGULATED BY A COMPLETE COMPLEMENT OF VIRAL CIS AND TRANS ACTING CONTROL ELEMENTS, SUCH THAT THE LEVEL OF EXTRACELLULAR ALKALINE PHOSPHATASE WILL SERVE AS AN INDICATOR OF VIRAL GAG, POL, AND ENV REGIONS SUBJECT TO NORMAL REGULATION BY THE VIRAL CODED TAT, REV, AND NEF GENE PRODUCTS. THE H9 CELL LINE WILL BE TRANSFECTED WITH THIS CONSTRUCT AND THE TRANSIENT EXPRESSION OF SPAP, P24, AND REVERSE TRANSCRIPTASE CHARACTERIZED. THE FEASIBILITY OF USING THE SPAP AS A MARKER TO DETECT ANTIVIRAL ACTIVITY WILL BE ASSESSED BY MONITORING THE EFFECT OF THREE ANTISENSE OLIGONUCLEOTIDES (ODNS) TARGETED TO CRITICAL SITES ON THE HIV GENOME IN THE TRANSFECTED H9 CELLS.