Design and cloning of an anti HIV-1 Rev hammerhead ribozyme.

The Human Immunodeficiency Virus (HIV) causes the Acquired Immunodeficiency Syndrome (AIDS). HIV is an RNA virus belonging to the Lentivirus group of retroviruses. Nine genes are expressed by the HIV genome. One of these genes, Rev (Regulator of expression of Viral protein) expresses a small protein that is responsible for regulating splicing during posttranscriptional modification of HIV mRNA. Rev expression results in generation of partially spliced and non-spliced mRNAs, which direct packaging and budding of viral progeny. This makes Rev essential to viral replication, and therefore a good target for anti viral reagents. Ribozymes are a potential means to inhibit viral replication by targeting and cleaving viral mRNAs in a sequence specific manner. The purpose of this experiment was to design and synthesize a catalytic hammerhead ribozyme targeted to the GUA target site located at nucleotide 8618 NL43 HIV Rev gene. A hammerhead ribozyme was designed based on the model of Haseloff and Gerlach (Nature 1988). This ribozyme consisted of flanking sequences complementary to Rev mRNA and the ribozyme catalytic core. Multiple copies of double stranded DNA (ds DNA) were generated by PCR using ribozyme specific primers and the synthesized ribozyme sequence. The ds DNA ribozyme was cloned into the pPCR-Script vector by blunt end ligation and transformed into XL-10 Gold Kan ultracompetent cells. Six colonies were picked for analysis, and plasmid DNAs were screened for the presence of the Rev ribozyme by PCR using a M13 primer pair. One clone that generated a 276 base pair fragment was sent for sequencing to verify the presence of the ribozyme. Future directions involve the design and synthesis of a non-catalytic hammerhead ribozyme targeted to the Rev gene at the nucleotide sequence 8618. * Supported by NIH Grant 1R15 GM66678-01