Lentiviral Vectors Expressing Chimeric NEDD4 Ubiquitin Ligases: An Innovative Approach for Interfering with Alpha-Synuclein Accumulation.

One of the main pathological features of Parkinson's disease (PD) is a diffuse accumulation of alpha-synuclein (aS) aggregates in neurons. The NEDD4 E3 Ub ligase promotes aS degradation by the endosomal-lysosomal route. Interestingly, NEDD4, as well as being a small molecule able to trigger its functions, is protective against human aS toxicity in evolutionary distant models. While pharmacological activation of E3 enzymes is not easy to achieve, their flexibility and the lack of "consensus" motifs for Ub-conjugation allow the development of engineered Ub-ligases, able to target proteins of interest. We developed lentiviral vectors, encoding well-characterized anti-human aS scFvs fused in frame to the NEDD4 catalytic domain (ubiquibodies), in order to target ubiquitinate aS. We demonstrate that, while all generated ubiquibodies bind to and ubiquitinate aS, the one directed against the non-amyloid component (NAC) of aS (Nac32HECT) affects aS's intracellular levels. Furthermore, Nac32HECT expression partially rescues aS's overexpression or mutation toxicity in neural stem cells. Overall, our data suggest that ubiquibodies, and Nac32HECT in particular, represent a valid platform for interfering with the effects of aS's accumulation and aggregation in neurons.

Targeting and Understanding HIV Latency: The CRISPR System against the Provirus.

The presence of latently infected cells and reservoirs in HIV-1 infected patients constitutes a significant obstacle to achieve a definitive cure. Despite the efforts dedicated to solve these issues, the mechanisms underlying viral latency are still under study. Thus, on the one hand, new strategies are needed to elucidate which factors are involved in latency establishment and maintenance. On the other hand, innovative therapeutic approaches aimed at eradicating HIV infection are explored. In this context, advances of the versatile CRISPR-Cas gene editing technology are extremely promising, by providing, among other advantages, the possibility to target the HIV-1 genome once integrated into cellular DNA (provirus) and/or host-specific genes involved in virus infection/latency. This system, up to now, has been employed with success in numerous in vitro and in vivo studies, highlighting its increasing significance in the field. In this review, we focus on the progresses made in the use of different CRISPR-Cas strategies to target the HIV-1 provirus, and we then discuss recent advancements in the use of CRISPR screens to elucidate the role of host-specific factors in viral latency.