Inhibition of Human Immunodeficiency Virus Type 1 Replication by Nuclear Chimeric Anti-HIV Ribozymes in a Human T Lymphoblastoid Cell Line

Abstract

Human immunodeficiency virus (HIV) infection represents one of the most challenging systems for gene therapy. Thanks to the extended knowledge of the molecular biology of the HIV life cycle, many different strategies have been developed including transdominant modifications of HIV proteins, RNA decoys, antisense RNA, ribozymes, and intracellular antibody fragments. In this paper, we have tested in a human T lymphoblastoid cell line the antiviral activity of ribozymes specifically designed to co-localize inside the nucleus with the Rev pre-mRNA before it is spliced and transported to the cytoplasm. This result was obtained by inserting the ribozyme in the spliceosomal U1 small nuclear RNA (snRNA) and in a derivative that has perfect complementarity with the 5′ splice site of the Rev pre-mRNA. These ribozymes were tested in human T cell clones and were shown to be very efficient in inhibiting viral replication. Not only were the p24 levels in the culture medium drastically reduced but so were the intracellular HIV transcripts. Control disabled ribozymes enabled us to show the specificity of the ribozyme activity. Therefore, these constructs have potential utility for gene therapy of HIV-1 infection.

Overview summary

Chimeric ribozymes, cloned inside the gene for the U1 small nuclear RNA, are efficiently expressed in a human T lymphoblastoid cell line. Stable clones transfected with anti-HIV ribozymes demonstrated significant resistance to human immunodeficiency virus type 1 (HIV-1) replication. Thus, intracellular immunization may be approachable using ex vivo transduction of human cells with anti-HIV U1-chimeric ribozymes in HIV-1-infected individuals.