High transdominant RevM10 protein levels are required to inhibit HIV-1 replication in cell lines and primary T cells: implication for gene therapy of AIDS

Abstract

Expression of antiviral genes in CD4⁺ T cells has been proposed as a strategy for gene therapy of AIDS. Over the past years, we and others have developed retroviral vectors encoding the RevM10 protein, a dominant-negative mutant of the HIV-1 Rev trans-activator protein. We could demonstrate gene transfer and inhibition of HIV-1 replication in cultured T cell lines and primary T cells. However, little is known about the levels of the antiviral protein required to achieve a therapeutic effect, particularly in primary cells. In this report, we compare different vector designs with regard to expression of the antiviral gene to develop an optimal vector for clinical applications. Our results demonstrate that intracellular steady-state RevM10 protein levels expressed from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV) or mouse embryonic stem cell virus (MESV) promoters located in the long terminal repeat (LTR) were uniformly higher than from internal promoters (eg CMV, PGK). Analysis of selected vectors in acutely and chronically HIV-infected cell lines suggested that threshold levels of RevM10 expression are required to achieve inhibition of HIV replication. LTR-driven RevM10 expression also yielded high steady-state protein levels in activated primary T cells resulting in inhibition of HIV replication, and there was no apparent difference between the MoMLV, MPSV and MESV-LTR vectors. However, RevM10 expression was down-regulated in resting primary cells and consequently anti-HIV efficacy was significantly reduced. Taken together, the data suggest that relatively high steady-state levels of RevM10 protein are required to achieve inhibition of HIV replication and that the MPSV- and MESV-derived retroviral vectors show no advantage over the MoMLV-based vectors for expression of anti-HIV genes in human T cells.