| Acquired immunodeficiency syndrome ( AIDS ) and human immunodeficiency virus (HIV) infection continue to be major global health concerns, with 1,000,000 Chinese citizens infected by the end of 2002. Although highly active antiretroviral therapy ( HAART ) has led to profound and prolonged reductions in circulating virus levels in many individuals, high cost, side effects and increasingly the emergence of multidrug-resistant virus strains limit the use of HAART. Gene therapy for HIV infection has made rapid progress in recent years. Several strategies have been permitted in clinical trial. However, the lack of efficient vectors and protocols for the delivery of therapeutic genes to target cells possible side effects of exogenous vectors and HIV escape mutations all limit their usefulness. So, the need for new antiviral therapies that target additional steps of the virus life cycle is pressing.The discovery of the principal HIV-1 coreceptor has significantly impacted our understanding of how HIV-1 infects its target cells and how this relates to viral pathogenesis. It has been confirmed that, to trigger the membrane fusion process that leads to viral entry, HIV-1 must first interact with CD4, then with a coreceptor. The discovery that chemokine receptors are co-receptors for HIV-1 has opened the door for a number of novel antiviral approaches. Chemokines as RANTES, MIP and SDF-1 have been utilized in anti-HIV gene therapy. A recent study by Chen SY et al mimicked the natural resistance of the individuals with the genetic CCR-5 defect by inactivating CCR-5 using a novel intracellular chemokine (intrakine) strategy. The CC-chemokine, RANTES, a ligand for CCR-5, was targeted to the ER lumen and found to prevent effectively the transport of newly synthesized CCR-5 to the cell surface. The lymphocytes expressing the CC-intrakine (RANTES-K) were resistant to M-tropic HIV-1 infection, while retaining normal cell functions. In an accompanied study, SDF-1 a, a ligand for CXCR-4, was also targeted to the ER to inactivate CXCR-4, and the viable lymphocytes expressing CXC-intrakine (SDF-K) were found to resist T-tropic HIV-1 infection. These results indicate the therapeutic application of this genetic intrakine strategy to control HIV-1 infection. However, the clinical facts below must be taken into consideration: ?Some species of HIV-1 viruses are dual-tropic, which replicate well both in T cell lines and primary macrophages by using CCR-5 and CXCR-4. ?T-tropic and M-tropic HIV-1 viruses can co-exist in the same HIV infected individual. (3) It is possible that use of a CCR5 antagonist may drive virus evolution from R5 to X4 phenotype. The progression to AIDS after HIV-1 infection is associated with a phenotypic shift from an M-tropic to a T-tropic virus. (4) Some viruses could even by-pass the utilization of CCR-5, as documented in individuals with a homozygous CCR-5 genetic defect who were infected with HIV-1. Thus, it is conceivable that inactivation of either M-tropic or T-tropic coreceptors alone may not besufficient to prevent HIV-1 infection and disease progression. Apparently, co-inactivation of both the principal M-tropic and T-tropic coreceptors would be a superior approach to control HIV-1 infection.In this study, a novel "intrakine" strategy was utilized to co-inactivate genetically both CCR5 and CXCR4 in HIV-1 target cells. The principle of co-inactivation of CCR5 and CXCR4 was illustrated by targeting the CC-intrakine and CXC-intrakine to the lumen of the endoplasmic reticulum for intracellular blockade of the transport of newly synthesized chemokine coreceptors to the cell surface. Firstly, we constructed bicistronic eukaryotic vector and retro viral vector, which harbor the genes of HIV-1 coreceptors' ligands桼ANTES and SDF-I. In order to examine co-expression of RANTES and SDF-1 by the bicistronic vector, several cell strains were transfected with various expression vectors DNA. Finally, Env-mediated syncytium formation, envelope-complementation assay and p24 detectio...
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