Antiviral activity and protection of cells against human immunodeficiency virus type-1 using an antisense oligodeoxyribonucleotide phosphorothioate complementary to the 5'-LTR region of the viral genome.
A COS-like monkey kidney cell line stably transfected with the plasmids pCMVgagpol-rre-r with the gag and pol genes, and pCMV rev with the rev gene of HIV-1 derived from the cDNA clone BH10, was used as a model for assessing the effectiveness of antisense (AS) constructs, A 20-mer oligodeoxyribonucleotide (oligo) phosphorothioate sequence (5'-CCG CCC CTC GCC TCT TGC CG) complementary to a portion of the 5'-long terminal repeat (5'-LTR) of the HIV-1 genome was tested for its inhibitory effects on the biologically important processes of HIV-1 replication and proliferation. We observed a concentration-dependent inhibition of HIV protein synthesis. Desitometric analysis of data from Western blot analysis showed sequence-specific and concentration-dependent oligo inhibition of p24 viral core antigen formation in the low-microM range. When lipofectin was used as a delivery vehicle, a markedly increased potentiation of the AS activity of the sequence was observed at a lower concentration (0.1 microM), following a 24-h preincubation. The AS construct specifically inhibited intracellular p24 production in chronically HIV-1-infected cells of lymphoid origin (H9/IIIB cells) by 95%, resulting in a 15-fold inhibitory effect relative to a similar sequence thiolated at only seven single-base positions. A concentration-dependent attenuation in the reverse transcriptase activity and a reduction in viral p24 level was observed in the culture supernatant of AS-pretreated HIV-1-infected phytohemagglutinin A-stimulated human cord blood mononuclear cells. Incubation of a HIV-1-infected lymphoid cell line with AS sequence resulted in a marked reduction in syncytium formation, and therefore protected cells from the cytopathic effects of the virus. Furthermore, the AS oligo did not appear to be cytotoxic in cell growth rate and colony-forming ability assays. The AS oligo described in this report is a useful new tool for the molecular analysis of HIV-1 gene expression and proliferation, and may have potential as a therapeutic agent.
Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.
M I
MI
Anazodo
H
H
Salomon
A D
AD
Friesen
M A
MA
Wainberg
J A
JA
Wright
D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D002522:Chlorocebus aethiops; D015967:Gene Expression Regulation, Viral; D015658:HIV Infections / Q000517:prevention & control*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology / Q000382:microbiology; D013873:Thionucleotides / Q000627:therapeutic use; D014779:Virus Replication
D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; D012194:RNA-Directed DNA Polymerase