RESUMO
BACKGROUND: Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, however, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. These preconditions have not yet been completely fulfilled and, thus, success of suicide approaches has been limited so far. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection. RESULTS: When tBid was introduced into the HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector pLRed(INS)2R, very efficient induction of apoptosis was observed within 24 hours, but only in the presence of both HIV-1 regulatory proteins Tat and Rev. Induction of apoptosis was not observed in their absence. Cells containing this vector rapidly died when transfected with plasmids containing full-length viral genomic DNA, completely eliminating the chance for HIV-1 replication. Viral replication was also strongly reduced when cells were infected with HIV-1 particles. CONCLUSIONS: This suicide vector has the potential to establish a safe and effective gene therapy approach to exclusively eliminate HIV-1 infected cells before infectious virus particles are released.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Genes Transgênicos Suicidas , HIV-1/fisiologia , Replicação Viral/genética , Clonagem Molecular , Citometria de Fluxo , Vetores Genéticos , HIV-1/genética , Células HeLa , Humanos , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
RNA interference is a powerful tool used to inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. Almost all HIV-1 genes have been targets for small interfering RNA (siRNA) molecules, and HIV-1 replication can be specifically and successfully inhibited by this technique. RNA interference has been proposed as an alternative strategy to inhibit replication of drug-resistant viruses that emerge during suboptimal antiretroviral therapy for HIV-1. To investigate specific inhibition of drug-resistant HIV-1 by RNA interference, we designed siRNA molecules that recognize codons 181-188 of the reverse transcriptase (RT) gene of wild-type HIV-1 and HIV-1 carrying the M184V mutation, which confers high-level resistance to the RT inhibitor lamivudine. Using viral variants with single point mutations at codon 184, we measured the impact of these mutations on virus replication. We have demonstrated that siRNA targeting either wild-type HIV-1 or M184V variants inhibits replication of the corresponding virus, but does not influence replication of virus with a mismatch in the targeted region. Combining two effective siRNAs did not show synergistic inhibitory effect on HIV-1 replication. However, a combination of lamivudine and siRNA-M184V was very effective in inhibiting replication of both wild-type and variant M184V viruses in mixed infection experiments. Taken together, these results demonstrate that RNA interference might be useful in the treatment of drug-resistant HIV-1 infection.
