Novel HIV-1 Knockdown Targets Identified by an Enriched Kinases/Phosphatases shRNA Library Using a Long-Term Iterative Screen in Jurkat T-Cells

HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies

Supplemental Calculations from Geometric decompositions of collective motion

Supplemental Calculations for ‘Geometric Decompositions of Collective Motion

"In vitro" methylation of GCGC sites of the human HLA-DR alpha gene by E. coli HhaI methylase: effects of chromatin proteins.

--

Oligonucleotidi sintetici in grado di indurre il differenziamento eritroide

Piccole molecole a doppio filamento di DNA possono essere utilizzate per la sottrazione di fattori trascrizionali nel modulare l'espressione dei geni globinic

Analysis of the human HLA-DRA gene upstream region: evidence for a stem-loop array directed by nuclear factors

Sequence analysis of the far-upstream region of the human HLA-DRA gene has revealed the presence of Y' and X' boxes, highly homologous to the well characterized Y and X boxes present within the proximal-promoter region. Comparison of Y, Y', X, and X' box sequences present within different class II MHC genes of different species demonstrates that these boxes are conserved during evolution, suggesting an important role in regulation of gene expression. The far-upstream region and the proximal promoter region of the class II MHC genes could be organized in secondary structures, as suggested for the EA gene, the murine counterpart of the human HLA-DRA gene. The essential feature of this model is a dimerization of the proteins binding to X and X' and/or Y and Y' boxes resulting in a loop-out of the intervening DNA and a rapprochement of the far-upstream and proximal-promoter regions, and consequently of any proteins binding to them. We set up an in vitro approach in order to determine whether proteins bound to sequences present within far-upstream and proximal-promoter regions of the human HLA-DRA gene could direct a secondary structure assembly of regulative regions. Moreover, by gel retardation and DNase I footprinting assays, we demonstrate that similar proteins bind to Y and Y' boxes and, among these proteins, NF-Y was unambiguously identified by antibody-super shift experiments. Taken together, the data presented in this paper provide evidence supporting the hypothesis that a stem-loop array of the 5'-upstream region of the human HLA-DRA gene could be directed by nuclear factors. In this manner, additional nuclear factors bound to the far region could be driven in close proximity of the transcription initiation site