Phosphorothioate Hammerhead Ribozymes Targeting a Conserved Sequence in the V3 Loop Region Inhibit HIV-1 Entry

Seven different phosphorothioate DNA-RNA chimeric hammerhead ribozymes (RzV3-nT, n = 1—7) targeted against the V3 loop region of HIV-1 were synthesized. Two of these, RzV3-1T and RzV3-3T, efficiently cleaved transcribed envelope RNA of HXB2 in vitro. The target sequence of RzV3-1T belongs to a conserved region and is completely identical in the HIV-1 HXB2, NL432, and ADA strains. Furthermore, RzV3-1T cleaved the envelope RNA of HIV-1 SF162 with a single base substitution in the distal site. U87 cells expressing CD4 and coreceptors were used as target cells for infections with the SF162 and NL432 strains. Replication of both the NL432 and SF162 strains in RzV3-1T-treated cells was significantly lower than that in control cultures. Envelope gene product formation was measured quantitatively with a single-cycle infection assay using pseudovirus generated from cotransfection with one vector containing a luciferase reporter gene and one vector containing the envelope gene of HXB2, SF162, or ADA. Production of ...