Comparative Analysis of Five Highly Conserved Target Sites Within the HIV-1 RNA for Their Susceptibility to Hammerhead Ribozyme-Mediated Cleavage In Vitro and In Vivo

Abstract

Moloney murine leukemia virus (MMLV)-derived pUCMoTiN-based retroviral vectors were engineered to allow constitutive and Tat (trans-activator of transcription)-inducible expression of five hammerhead ribozymes targeted against highly conserved sequences within the group antigen (Gag), protease (Pro), reverse transcriptase (RT), tat, and envelope (Env) coding regions of human immunodeficiency virus type-1 (HIV-1) RNA. Amphotropic retroviral vector particles were used to infect a human CD4+ lymphocyte-derived MT₄ cell line. The pool of stable MT₄ transductants expressing these ribozymes were each tested for their susceptibility to HIV-1 infection. Rz_Tat conferred no protection to MT₄ cells. Rz_Gag and Rz_RT completely inhibited virus multiplication for 6 days. Rz_Pro and Rz_Env conferred the best protection, as they completely inhibited virus production for 12 and 15 days, respectively. No correlation was found between the degree of HIV-1 resistance conferred and the ability of these ribozymes to cleave their target RNA in vitro. From Rz_Pro-expressing HIV-1-infected cells following virus escape, Rz_Pro and target RNA sequences were amplified and checked for cleavage in vitro. The ribozyme expressed in these cells was shown to cleave the corresponding target RNA. Thus, a mutation in the ribozyme or target RNA does not seem to be the mechanism underlying virus escape.