Ribozyme-mediated suppression of Moloney murine leukemia virus and human immunodeficiency virus type I replication in permissive cell lines.
Several hammerhead ribozymes targeted to different sites within the retroviral packaging (psi) sequences of the Moloney murine leukemia virus (Mo-MLV) and the human immunodeficiency virus type 1 (HIV-1) were designed and shown to cleave target RNA in vitro at the chosen sites. The engineered ribozymes, as well as antisense sequence complementary to the Mo-MLV psi packaging region, were cloned into the 3' untranslated region of the neomycin-resistance gene (neo). This was coupled to the simian virus 40 early promoter within the pSV2neo vector. For the ribozymes against the Mo-MLV psi site, the constructs were transfected into Mo-MLV-infected and virus-producing mouse NIH 3T3 cells. With the exception of one of the single ribozymes (the one least effective in cutting target RNA in vitro), all of the constructs effectively (70-80%) suppressed retrovirus production. These results demonstrate a direct correlation between in vitro cleavage and in vivo ribozyme-mediated virus suppression. In addition, a ribozyme targeted to the HIV-1 psi packaging site was engineered into the same vector and transfected into the human T-cell line SupT1. The transfectants were cloned and then challenged with HIV-1. When compared to vector-transfected control cells, a significant reduction in HIV-1 production was observed as measured by p24 and syncytia formation assays. This study demonstrates a feasible approach to the suppression of retrovirus replication by targeting the psi packaging site with hammerhead ribozymes.
Sun
L Q
LQ
R.W. Johnson Pharmaceutical Research Institute-Sydney, New South Wales, Australia.
Warrilow
D
D
Wang
L
L
Witherington
C
C
Macpherson
J
J
Symonds
G
G
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005822:Genetic Vectors; D016679:Genome, Viral; D015726:Giant Cells; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000502:physiology*; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000378:metabolism*; D012367:RNA, Viral / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D010948:Viral Plaque Assay; D014779:Virus Replication
D016337:RNA, Catalytic; D012367:RNA, Viral