title: Rev-binding aptamer and CMV promoter act as decoys to inhibit HIV replication.
abstract: We examined whether the antiviral effect of an HIV-1 Rev-binding aptamer [RBE(apt)] could be enhanced by a ribozyme directed against the HIV-1 env gene, and whether the antiviral activity was affected by different promoters. The efficacy of the aptamer and ribozyme DNAs was tested in HeLa cells co-transfected with the HIV-1 proviral clones, HXBDeltaBgl or pNL4-3, using transferrin-lipoplexes. The RBE(apt) and anti-env ribozyme genes were inserted into the pTZU6+27 plasmid, or constructed under the control of the human cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters. The parental vector plasmids were used as controls. Co-transfection of the pTZU6+27 RBE(apt) plasmid with HXBDeltaBgl, or pNL4-3, at a weight ratio of 5:1, inhibited p24 production by 70 and 45%, respectively. The RSV RBE(apt) plasmid co-transfected with either HIV clone, at the same weight ratio, reduced viral production by 88%. The addition of the anti-env ribozyme to the RSV RBE(apt) did not enhance its antiviral activity. When the constructs were under the control of the CMV promoter, the expression of the HIV plasmids was very low and was independent of the presence of the RBE(apt). Thus, the effect of the RBE(apt) was strongly dependent on the promoter of the tested construct. The anti-HIV activity of the CMV RBE(apt) construct was non-specific, because co-transfection with either pCMV. SPORT-betagal or pCMVlacZ significantly suppressed HIV production from the HIV proviral clones. The reduction in p24 could not be attributed to the non-specific toxicity of the transfection procedure. Transfection of acutely HIV-infected HeLa-CD4 cells with pCMV.SPORT-betagal reduced the p24 level by 35%, while the expression of the U6 RBE(apt) did not affect p24 production. The suppression of HIV production from the HIV proviral clones by the CMV promoter constructs in the co-transfection assays may be explained by competition for transcription factors (TFs) between HIV and CMV promoters. This observation points to the potential for misleading results in co-transfections involving CMV constructs and HIV.
authors.affiliation: Department of Microbiology, School of Dentistry, University of the Pacific, San Francisco, CA 94115, USA. kkonopka@sf.uop.edu
authors.forename: K
authors.initials: K
authors.lastname: Konopka
authors.forename: N S
authors.initials: NS
authors.lastname: Lee
authors.forename: J
authors.initials: J
authors.lastname: Rossi
authors.forename: N
authors.initials: N
authors.lastname: Düzgüneş
mesh_terms: D019380:Anti-HIV Agents / Q000378:metabolism; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics*; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D015686:Gene Products, env / Q000378:metabolism; D015694:Gene Products, rev / Q000378:metabolism*; D016655:HIV Core Protein p24 / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D014779:Virus Replication / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus
chemical_list: D019380:Anti-HIV Agents; D004274:DNA, Recombinant; D015686:Gene Products, env; D015694:Gene Products, rev; D016655:HIV Core Protein p24; D009841:Oligonucleotides; D016337:RNA, Catalytic; D011993:Recombinant Fusion Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D008156:Luciferases; D001616:beta-Galactosidase