Intracellular inhibition of HIV-1 replication using a dual protein- and RNA-based strategy

Exporting unspliced human immunodeficiency virus type 1 (HIV-1) RNA from the nucleus to the cytoplasm, through an interaction between the viral regulatory Rev protein and Rev response element (RRE) RNA, is a critical step in the HIV-1 life-cycle. Disruption of either Rev or the RRE will completely inhibit HIV-1 replication. As such, a strategy for somatic gene therapy to treat HIV-1 infection by intracellular expression of an anti-HIV-1 Rev single chain variable fragment (SFv) and a ribozyme which specifically targets the RRE was developed. The anti-Rev D8SFv, which specifically targets the Rev activation domain, may be a key component of combination intracellular immunization, as it has been previously shown to potently inhibit Rev function, thereby inhibiting viral replication. In the present studies, different HIV-1 RRE region-specific hammerhead ribozymes were constructed and their anti-HIV-1 replication effects were assayed in diverse RNA polymerase (pol) II and III promoters and vector systems in cell culture. Utilizing this combination of an SFv and a ribozyme as a dual strategy to block HIV-1 replication, both at the protein and RNA level, data from these studies demonstrated that potent inhibition of HIV-1 replication can be achieved via this approach. Combination gene therapies hold promise, analogous to combination chemotherapeutic regimens, for the in vivo treatment of HIV-1 infections.