A chimeric human immunodeficiency virus type 1 (HIV-1) minimal Rev response element-ribozyme molecule exhibits dual antiviral function and inhibits cell-cell transmission of HIV-1.
We have previously shown that hairpin ribozymes targeting the human immunodeficiency virus (HIV) genome can effectively inhibit virus replication in a variety of primary and cultured hematopoietic cells. To further increase antiviral potency and minimize the chance of viral resistance, we have now cloned the stem-loop II sequences of the HIV type 1 Rev response element into ribozyme transcription cassettes. Fusion RNA molecules were shown to function both as RNA decoys and ribozymes. Stable Molt-4/8 cell lines expressing fusion RNA of stem-loop II and a ribozyme directed at the HIV-type 1 U5 sequence (MSLMJT) or its disabled counterpart (MSLdMJT) were generated. The expression of fusion RNA was persistent for at least 6 months without apparent cytotoxicity. When virus inhibition was examined after the cocultivation of transduced cells with chronically infected Jurkat cells, much greater protection was observed in MSLMJT cells than in MSLdMJT or MMJT (expressing only the ribozyme) cells. Furthermore, to specifically compare the ribozyme activities in various transduced cells, we determined the quantitative levels of proviral DNA in the first round of virus replication (7 h after infection with HXB2). By competitive PCR, the proviral DNA levels in MSLMJT and MMJT cells were found to be reduced to 1/7 and 1/3, respectively, compared with those in MSLdMJT and MdMJT cells. These results suggest not only that the greater inhibition afforded by this fusion RNA was due to its function both as decoy and ribozyme but also that the ribozyme activity may be facilitated.
Yamada
O
O
Department of Medicine, University of California, San Diego, La Jolla 92093-0665, USA.
Kraus
G
G
Luznik
L
L
Yu
M
M
Wong-Staal
F
F
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral; D015694:Gene Products, rev / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000235:genetics / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects; D054321:rev Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D015694:Gene Products, rev; D016337:RNA, Catalytic; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus