Inhibition of HIV-1 replication by RNA targeted against the LTR region.
OBJECTIVE
The use of small RNA molecules able to effect gene inactivation has emerged as a powerful method of gene therapy. These small inhibitory RNAs are widely used for silencing malignant cellular and viral genes. We have assayed a series of inhibitory RNAs named catalytic antisense RNAs, consisting of a catalytic domain, hairpin or hammerhead ribozyme, and an antisense domain. The aim of the present study was to evaluate the effect of these inhibitory RNAs on HIV-1 replication.


METHODS
A series of expression vectors has been constructed for the intracellular synthesis of inhibitory RNAs, differing in the promoter that drives their synthesis. These inhibitory RNAs were designed to act at two possible cleavage sites in the long terminal repeat (LTR) region and the TAR domain was chosen as a target for the antisense domain. We have evaluated the effects of different inhibitory RNAs in HIV replication via changes in p24 antigen levels. Mobility shift assays have been used to check the binding capacity of inhibitory RNAs.


RESULTS
Catalytic antisense RNA designed to target the LTR region of HIV-1 inhibited viral replication in an eukaryotic cell environment by more than 90%. The conventional hairpin and hammerhead ribozymes, however, failed to inhibit viral replication.


CONCLUSIONS
The data provide preliminary evidence of a new class of inhibitory RNAs that can be used to block HIV replication. The results clearly show the importance of the ex vivo antisense effect in the inhibition achieved. A good correlation was found between the in vitro binding efficiency of the inhibitor RNA to the HIV-1 LTR and the inhibition of viral replication.
Puerta-Fernández
Elena
E
Instituto de Parasitología y Biomedicina López-Neyra, CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla, 18100 Granada, Spain.
Barroso-del Jesus
Alicia
A
Romero-López
Cristina
C
Tapia
Natalia
N
Martínez
Miguel Angel
MA
Berzal-Herranz
Alfredo
A
D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D016655:HIV Core Protein p24 / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D016372:RNA, Antisense / Q000096:biosynthesis / Q000235:genetics; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000235:genetics*
D016655:HIV Core Protein p24; D016372:RNA, Antisense; D016337:RNA, Catalytic