227 Lentiviral Vectors Interfering with CD4 Down-modulation as an Anti-HIV Approach B. Gröschel*, M.J. Cortés, E. Argañaraz, J. Lama Univ of California at San Diego, La Jolla
Background: Physiological levels of cell-surface CD4 interfere with HIV-1 replication in T-cells by a mechanism that inhibits envelope incorporation into viral membranes. It suggests that CD4 down-modulation in HIV-1 infected cells is required for the production of infectious virus particles. Three (3) viral gene products (Nef, Vpu, and Env) participate in the removal of CD4 molecules from the cell surface. Nef and Vpu bind CD4 through its cytoplasmic domain and down-regulate its expression by targeting CD4 to intracellular pathways of degradation or by blocking the transport of CD4 from the ER to the cell surface. Despite the importance of HIV-mediated CD4 down-modulation in the virus life cycle, to date this function has not been the target for antiviral approaches. In this study we have engineered different lentiviral vectors that deliver modified CD4 genes into HIV-1 target cells and interfere with virus infectivity.
Methods: HIV-based lentiviral vectors were constructed that were able to express either recombinant CD4 wild-type protein, CD4 lacking the cytoplasmic domain, a fusion protein of CD4 and HIV-1 matrix or GFP as control. Viral particles expressing modified CD4 molecules or GFP were produced by co-transfection of 293 T-cells in the presence of a packaging construct, encoding HIV structural proteins and a plasmid encoding the VSV g protein, which allows the entry into cells. Viral particles were quantify by p24 antigen ELISA. Transduction efficiency of viral particles expressed as CD4 expression was tested by FACS analysis. Molecular sizes of modified CD4 proteins were determined by SDS-PAGE and Western blot analysis.
Results: Viral particles delivering modified CD4 genes were able to transduce 293 T and Jurkat T-cells efficiently and modified CD4 molecules were expressed in the right molecular size. CD4 molecules lacking their cytoplasmic domain were resistant to the Nef-mediated CD4 down-modulation process. Moreover, virus particles produced from HIV-1 infected cells expressing modified CD4 molecules were drastically less infectious than those produced in cells expressing CD4 wild-type proteins.
Conclusions: Our results show that expression of CD4 molecules insensitive to the action of Nef interferes with the infectivity of released HIV-1 particles and suggests that interfering with the HIV-1 mediated CD4 down-modulation process might constitute the rationale for a novel anti-HIV-1 strategy.
