Phosphorothioate hammerhead ribozymes targeting a conserved sequence in the V3 loop region inhibit HIV-1 entry

Seven different phosphorothioate DNA-RNA chimeric hammerhead ribozymes (RzV 3-nT, n = 1-7) targeted against the V3 loop region of HIV-1 were synthesize d. Two of these, RzV3-1T and RzV3-3T, efficiently cleaved transcribed envel ope RNA of HXB2 in vitro. The target sequence of RzV3-1T belongs to a conse rved region and is completely identical in the HIV-1 HXB2, NL432, and ADA s trains. 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 NL4 32 strains. Replication of both the NL432 and SF162 strains in RzV3-1T-trea ted cells was significantly lower than that in control cultures. Envelope g ene product formation was measured quantitatively with a single-cycle infec tion assay using pseudovirus generated from cotransfection with one vector containing a luciferase reporter gene and one vector containing the envelop e gene of HXB2, SF162, or ADA, Production of pseudovirus in RzV3-1T-treated cells led to a marked (93% or 87%) inhibition of envelope-mediated entry o f resultant HXB2-derived or ADA-derived pseudotype virions, respectively, a nd a moderate (44%) inhibition was seen for SF162-derived pseudotype virion s. Thus, an efficient, stable ribozyme against a functionally important reg ion of HIV-1 was identified by evaluating its activities in vitro and in vi vo. This ribozyme may be useful for control of HIV-1 infection.