Design and cloning of a hammerhead ribozyme targeted to HIV-1 VPU.

The Human Immunodeficiency Virus is a retrovirus responsible for the Acquired Immunodeficiency Syndrome (AIDS), a devastating epidemic that affects millions worldwide. HIV causes the gradual deterioration of immune function by destroying CD4+ T lymphocytes. AIDS occurs due to the loss of these cells accompanied by an increased susceptibility to infections and cancers. Vpu (Viral Protein U) is one of fifteen distinct proteins encoded by the HIV-1 genome. Vpu promotes degradation of the CD4 receptor and may be a good target for antiviral reagents such as ribozymes. Ribozymes are RNA molecules that catalyze sequence specific cleavage of RNA substrates in a sequence specific manner. Hammerhead ribozymes, as based on the Haseloff and Gerlach model (Nature, 1988), consist of a catalytic core made up of a highly conserved sequence of 24 nucleotides that cleave substrate RNAs. The catalytic core is made sequence specific by two flanking regions that will pair in a complementary manner with nucleotides 5' and 3' of the target site. This project involved the design and synthesis of a catalytic hammerhead ribozyme targeted to the GUA sequence at nucleotide 6134 of the HIV-1 NL43 Vpu gene. The ribozyme template was designed based on the Haseloff and Gerlach model and double stranded ribozyme DNA was generated PCR using ribozyme specific primers. Double stranded DNA was cloned into pSTBlue-1 by blunt end ligation and transformed into Nova Blue ultracompetent cells. Five experimental colonies and one control colony were picked and miniprep DNA was prepared for each. The presence of the cloned ribozyme was determined by PCR using a T7 and SP6 primer pair. Clone 2 generated a PCR fragment of 227bp and was selected for direct sequencing to verify the presence of the catalytic Vpu ribozyme. Future directions include the design and synthesis of a non-catalytic hammerhead ribozyme targeted to Vpu 6134 for use as a control. * Supported by NIH Grant 1R15 GM66678-01