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 containing 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 infectious clone SF2 contains a single nucleotide substitution at the cleavage site (from N*GUC to N*UUC). Cells stably expressing the ribozyme or its disabled counterpart were challenged with HXB2 or SF2 and the amount of p24 antigen produced was monitored. While this ribozyme was effective 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 ribozyme, which functions essentially as an antisense molecule. Expression of the ribozyme was comparable in HXB2- or SF2-infected cells as detected by reverse transcription-polymerase chain reaction. These data provide in vivo evidence that the antiviral activity of the hairpin ribozyme is strictly dependent on the presence of the cleavage site in the target RNA and supports the conclusion that the ribozyme acts as catalytic RNA rather than as antisense RNA in vivo.
Yamada
O
O
Department of Medicine and Biology, University of California, San Diego 92093-0665.
Kraus
G
G
Leavitt
M C
MC
Yu
M
M
Wong-Staal
F
F
D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology / Q000821:virology*; D002460:Cell Line; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D013379:Substrate Specificity; D014779:Virus Replication
D016337:RNA, Catalytic; D012367:RNA, Viral