Impact of the Ku complex on HIV-1 expression and latency.

Ku, a cellular complex required for human cell survival and involved in double strand break DNA repair and multiple other cellular processes, may modulate retroviral multiplication, although the precise mechanism through which it acts is still controversial. Recently, Ku was identified as a possible anti-human immunodeficiency virus type 1 (HIV-1) target in human cells, in two global approaches. Here we investigated the role of Ku on the HIV-1 replication cycle by analyzing the expression level of a panel of non-replicative lentiviral vectors expressing the green fluorescent protein in human colorectal carcinoma HCT 116 cells, stably or transiently depleted of Ku. We found that in this cellular model the depletion of Ku did not affect the efficiency of (pre-)integrative steps but decreased the early HIV-1 expression by acting at the transcriptional level. This negative effect was specific of the HIV-1 promoter, required the obligatory step of viral DNA integration and was reversed by transient depletion of p53. We also provided evidence on a direct binding of Ku to HIV-1 LTR in transduced cells. Ku not only promotes the early transcription from the HIV-1 promoter, but also limits the constitution of viral latency. Moreover, in the presence of a normal level of Ku, HIV-1 expression was gradually lost over time, likely due to the counter-selection of HIV-1-expressing cells. On the contrary, the reactivation of transgene expression from HIV-1 by means of trichostatin A- or tumor necrosis factor α-administration was enhanced under condition of Ku haplodepletion, suggesting a phenomenon of provirus latency. These observations plead in favor of the hypothesis that Ku has an impact on HIV-1 expression and latency at early- and mid-time after integration.

3' self-inactivating long terminal repeat inserts for the modulation of transgene expression from lentiviral vectors.

Gene transfer for research or gene therapy requires the design of vectors that allow for adequate and safe transgene expression. Current methods to modulate the safety and expression profile of retroviral vectors can involve the insertion of insulators or scaffold/matrix-attachment regions in self-inactivating long terminal repeats (SIN-LTRs). Here, we generated a set of lentiviral vectors (with internal CMV or PGK promoter) in which we inserted (at the level of SIN-LTRs) sequences of avian (i.e., chicken hypersensitive site-4, cHS4), human (i.e., putative insulator and desert sequence), or bacterial origin. We characterized them with respect to viral titer, integration, transduction efficiency and transgene expression levels, in both integrase-proficient and -deficient contexts. We found that the cHS4 insulator enhanced transgene expression by a factor of 1.5 only when cloned in the antisense orientation. On the other hand, cHS4 in the sense orientation as well as all other inserts decreased transgene expression. This attenuation phenomenon persisted over long periods of time and did not correspond to extinction or variegation. Decreased transgene expression was associated with lower mRNA levels, yet RNA stability was not affected. Insertions within the SIN-LTRs may negatively affect transgene transcription in a direct fashion through topological rearrangements. The lentiviral vectors that we generated constitute valuable genetic tools for manipulating the level of transgene expression. Moreover, this study demonstrates that SIN-LTR inserts can decrease transgene expression, a phenomenon that might be overcome by modifying insert orientation, thereby highlighting the importance of careful vector design for gene therapy.