AEGiS-11IAC: Development of novel anti-HIV agents that interfere with a ribosomal frameshifting event required for the production of viral enzymes.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Development of novel anti-HIV agents that interfere with a ribosomal frameshifting event required for the production of viral enzymes.

Int Conf AIDS 1996 Jul 7-12; 11:10 (abstract no. We.A.275)
Melancon P, Payant C, Yao XJ, Cohen EA, Brakier-Gingras L; Biochimie, Univ. Montreal, Montreal, PQ, Canada. Fax: 514-343-2210. E-mail: gingras@bch.umontreal.ca.

OBJECTIVE: The synthesis of the viral enzymes of the human immunodeficiency virus (HIV-1) requires a -1 ribosomal frameshift, when ribosomes of infected cells translate the viral messenger. Our goal is to interfere with this frameshift, so as to inhibit viral assembly and proliferation.

METHODS: The ribosomal RNA (rRNA) is involved in the frameshifting event. We decided to target specific portions of this rRNA with complementary oligodeoxynucleotides (antisense agents) that, by binding transiently to the rRNA, might interfere with the ribosomal frameshift. The shifty region of HIV-1 was inserted in the 5'end region of a reporter gene, the bacterial chloramphenicolacetyltransferase. The construction was such that this gene was expressed only by the ribosomes that shifted the reading frame. The messenger RNA corresponding to the reporter gene was translated in vitro in a reticulocyte lysate, in the presence of different oligomers targeted to the rRNA. The engineered reporter gene cloned in a mammalian expression vector was also transfected into HeLa cells, in the presence of oligomers that were modified in the sugar moiety (2'O-methyl or 2'O-allyl), so as to avoid degradation by intracellular nucleases.

RESULTS: Two 12-mer oligomers targeted to the 530-like region of the rRNA of the small ribosomal subunit efficiently decreased (by more than two-fold) ribosomal frameshifting both in vitro and in the HeLa cells, without significantly affecting the normal function of the ribosomes. Preliminary results with HeLa-CD4+ cells transfected with HIV-1 DNA or with T-lymphocytes (MT-4 cell line) infected with HIV confirmed that the two oligomers interfere with HIV proliferation, as assessed by standard assays such as syncitia formation and reverse transcriptase activity.

CONCLUSION: We demonstrated that antisense oligomers targeted to a specific portion of the rRNA can interfere with HIV proliferation, by decreasing a ribosomal frameshift that is indispensable for virus development. Since the target is the host ribosomes, which are independent of virus mutability, the risk of emergence of drug-resistant viral variants is extremely unlikely with a therapeutic approach using these antisense oligomers (supported by MRC-PMAC and Theratechnologies).

Keywords: AEGIS, Frameshifting, Ribosomal, Anti-HIV Agents, HIV-1, RNA-Directed DNA Polymerase, HIV, Ribosomes, Reverse Transcriptase Inhibitors, HIV Protease Inhibitors, Virus Assembly, HIV Infections, Ribonuclease H, Calf Thymus, Oligonucleotides, Antisense, Human, In Vitro, enzymology, growth & development, virology, genetics, ICA11

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WeA275