title: Use of the bovine leukaemia virus LTR U3 promoter for expressing antisense antiviral RNAs and competitive inhibition of viral infection in cell culture.
abstract: Use of viral inducible promoters which can be activated by virus-specific transactivator proteins to drive expression of antisense (as)RNA genes appears to be an attractive approach to inhibit virus infections in vivo. To this end, we have constructed an asRNA gene expressed from the bovine leukaemia virus (BLV) U3 promoter that is complementary to the R-U5 region of the BLV genome. This is the region that is most susceptible to inhibition by asRNA. With plasmid pLU, which expresses the asRNA gene under the control of the BLV U3 promoter, 75% inhibition of virus replication was attained in CC81 cells (the molar ratio of pLU DNA over BLV proviral DNA in the transfection mixture was 5:1). Plasmid pLT, which contains only the BLV U3 promoter without any asRNA-coding region, also efficiently (up to 60%) inhibited virus replication when cotransfected with BLV proviral DNA at a ratio of 20:1. It was suggested that competition between functional and 'empty' viral promoters for the viral transactivator protein p38tax could account for this inhibition. An immunoblotting assay showed that in the presence of nuclear extracts from CC81 cells exogenous BLV p38tax specifically associates with its responsive sequence located in the BLV U3 promoter. Moreover, the additional expression of p38tax in CC81 cells abolishes the inhibitory effect of the empty viral promoter. These observations suggest a new mechanism of BLV inhibition caused, most probably, by sequestering of the viral transactivator protein.
authors.affiliation: Department of Genetic Engineering, Institute of Agricultural Biotechnology, Moscow, Russia.
authors.forename: D
authors.initials: D
authors.lastname: Shayakhmetov
authors.forename: D
authors.initials: D
authors.lastname: Kovalenko
authors.forename: G
authors.initials: G
authors.lastname: Yurov
authors.forename: A
authors.initials: A
authors.lastname: Borisenko
authors.forename: T
authors.initials: T
authors.lastname: Tikchonenko
mesh_terms: D000818:Animals; D002415:Cats; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D016356:Gene Products, tax / Q000096:biosynthesis / Q000235:genetics; D015726:Giant Cells; D001909:Leukemia Virus, Bovine / Q000235:genetics / Q000502:physiology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics / Q000502:physiology; D015347:RNA Probes; D016372:RNA, Antisense / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D015682:Retroviridae Proteins, Oncogenic / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D016356:Gene Products, tax; D015347:RNA Probes; D016372:RNA, Antisense; D012367:RNA, Viral; D011994:Recombinant Proteins; D015682:Retroviridae Proteins, Oncogenic; C059786:transactivator protein p38(tax)