No abstract

APOBEC3 proteins can inhibit human immunodeficiency virus type 1 (HIV-1) replication by inducing G-to-A mutations in newly synthesized viral DNA. However, HIV-1 is able to overcome the antiretroviral activity of some of those enzymes by the viral protein Vif. We investigated the impact of different processivities of HIV-1 reverse transcriptases (RT) on the frequencies of G-to-A mutations introduced by APOBEC3 proteins. Wild-type RT or the M184V, M184I, and K65R؉M184V RT variants, which are increasingly impaired in their processivities, were used in the context of a vif-deficient molecular HIV-1 clone to infect H9 cells and peripheral blood mononuclear cells (PBMCs). After two rounds of infection, a part of the HIV-1 env gene was amplified, cloned, and sequenced. The M184V mutation led to G-to-A mutation frequencies that were similar to those of the wild-type RT in H9 cells and PBMCs. The frequencies of G-to-A mutations were increased after infection with the M184I virus variant. This effect was augmented when using the K65R؉M184V virus variant (P < 0.001). Overall, the G-to-A mutation frequencies were lower in PBMCs than in H9 cells. Remarkably, 38% ؎ 18% (mean ؎ standard deviation) of the env clones derived from PBMCs did not harbor any G-to-A mutation. This was rarely observed in H9 cells (3% ؎ 3%). Our data imply that the frequency of G-to-A mutations induced by APOBEC3 proteins can be influenced by the processivities of HIV-1 RT variants. The high number of nonmutated clones derived from PBMCs leads to several hypotheses, including that additional antiretroviral mechanisms of APOBEC3 proteins other than their deamination activity might be involved in the inhibition of vif-deficient viruses.

BackgroundRegulated 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.ResultsWhen 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.ConclusionsThis 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.