Conservation of a Hairpin Ribozyme Sequence in HIV-1 Is Required for Efficient Viral Replication

We have previously described a hairpin ribozyme that targets a highly conserved sequence in the U5 region of HIV-1. To determine if escape mutations would compromise virus replication, we introduced critical mutations into the ribozyme target site of an infectious molecular clone of HIV-1MN. HIV-1 MN_Ahas a substitution of A for G immediately 3′ to the cleavage site and HIV-1 MN_GChas two substitutions in the flanking sequences that are complementary to the ribozyme.In vitrostudies confirmed that neither the MN_A- nor the MN_GC-mutated target sequence was cleaved by the ribozyme, and furthermore, the MN_GC-mutated target sequence failed to bind the ribozyme. Compensatory GC substitutions in the substrate recognition domain of the ribozyme resulted in a switch of binding and cleavage specificity. Replication of both the MN_Aand MN_GCmutant viruses was initially two to three logs lower than that of wild-type virus, but after 3 weeks, virus production rose sharply in both cultures. Nucleotide sequence of RT-PCR-amplified viral sequences obtained from virus produced at later time points revealed complete reversion of MN_Aor partial reversion of MN_GCto wild-type genotypes. No additional mutations within the ribozyme target sequence were observed. These results indicate that mutations in this conserved ribozyme target sequence led to significant attenuation of HIV-1MN.