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 MNA has a substitution of A for G immediately 3' to the cleavage site and HIV-1 MNGC has two substitutions in the flanking sequences that are complementary to the ribozyme. In vitro studies confirmed that neither the MNA-nor the MNGC-mutated target sequence was cleaved by the ribozyme, and furthermore, the MNGC-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 MNA and MNGC mutant 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 MNA or partial reversion of MNGC to 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.
Yamada
O
O
Department of Medicine, University of California at San Diego, La Jolla 92093-0665, USA.
Kraus
G
G
Sargueil
B
B
Yu
Q
Q
Burke
J M
JM
Wong-Staal
F
F
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017124:Conserved Sequence; D004279:DNA, Viral; D016655:HIV Core Protein p24 / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D004279:DNA, Viral; D016655:HIV Core Protein p24; D016337:RNA, Catalytic; D012367:RNA, Viral