ACTIVITY AND CLEAVAGE SITE-SPECIFICITY OF AN ANTI-HIV-1 HAIRPIN RIBOZYME IN HUMAN T-CELLS

Human CD4+ T cells (Molt-4) were transduced with retroviral vectors co ntaining a hairpin ribozyme which targets the rev/env coding region of HIV-1 RNA (HXB2: 8629-8644). This target sequence is conserved among many HIV-1 clones, including the prototype virus HXB2, but the infecti ous clone SF2 contains a single nucleotide substitution at the cleavag e site (from NGUC to N*UUC). Cells stably expressing the ribozyme or its disabled counterpart were challenged with HXB2 or SF2 and the amou nt of p24 antigen produced was monitored. While this ribozyme was effe ctive in inhibiting the replication of HXB2 in Molt 4 cells, it showed only marginal inhibitory effect on SF2 replication. The same level of virus production was observed with cells transduced by the disabled r ibozyme, which functions essentially as an antisense molecule. Express ion of the ribozyme was comparable in HXB2- or SF2-infected cells as d etected by reverse transcription-polymerase chain reaction. These data provide in vivo evidence that the antiviral activity of the hairpin r ibozyme is strictly dependent on the presence of the cleavage site in the target RNA and supports the conclusion that the ribozyme acts as c atalytic RNA rather than as antisense RNA in vivo. (C) 1994 Academic P ress, Inc.