Inhibition of HIV-1 replication by an HIV-1 dependent ribozyme expression vector with the Cre/loxP (ON/OFF) system.
Antiviral strategies to inhibit HIV-1 replication have included the generation of gene products that provide the intracellular inhibition of an essential viral protein or RNA. When used in conjunction with the HIV-1 long terminal repeat (LTR), an inducible promoter dependent on the virus-encoded trans-activator (tat), relatively high background activity is still observed in the absence of tat (Caruso & Klatzmann, 1992; Dinges et al., 1995). In order to circumvent this problem, we used the Cre/loxP (ON/OFF) recombination system as a tool for our investigation. In the present study, we constructed a loxP-cassette vector with the ribozyme (Rz) expression portion under the control of the tRNAi(Met) promoter between two loxP sequences (plox-Rz-U5). We also constructed an HIV-1 LTR promoter-driven Cre recombinase gene (pLTR-Cre). These vectors were triple-transfected into HeLa CD4 cells with the HIV-1 pseudotype viral expression vector. Basal activity was not detectable before HIV-1 infection. The LTR-dependent Cre protein product in HIV-1 infected HeLa CD4 cells expressed the ribozyme by inducing loxP homologous recombination, which strongly inhibited the HIV-1 gene expression. These results demonstrate the potential of an anti-ribozyme with the Cre/loxP system for controlling HIV-1 infection via gene therapy.
Habu
Yuichiro
Y
Department of Industrial Chemistry, Chiba Institute of Technology, Narashino, Chiba, Japan.
Miyano-Kurosaki
Naoko
N
Nagawa
Takashi
T
Matsumoto
Norihiko
N
Takeuchi
Hiroaki
H
Takaku
Hiroshi
H
D001287:Attachment Sites, Microbiological / Q000235:genetics*; D005786:Gene Expression Regulation; D015967:Gene Expression Regulation, Viral; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D016337:RNA, Catalytic; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D012367:RNA, Viral; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases