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

Insights into household transmission of SARS-CoV-2 from a population-based serological survey

Understanding the risk of infection from household- and community-exposures and the transmissibility of asymptomatic infections is critical to SARS-CoV-2 control. Limited previous evidence is based primarily on virologic testing, which disproportionately misses mild and asymptomatic infections. Serologic measures are more likely to capture all previously infected individuals. We apply household transmission models to data from a cross-sectional, household-based population serosurvey of 4,534 people ≥5 years from 2,267 households enrolled April-June 2020 in Geneva, Switzerland. We found that the risk of infection from exposure to a single infected household member aged ≥5 years (17.3%,13.7-21.7) was more than three-times that of extra-household exposures over the first pandemic wave (5.1%,4.5-5.8). Young children had a lower risk of infection from household members. Working-age adults had the highest extra-household infection risk. Seropositive asymptomatic household members had 69.4% lower odds (95%CrI,31.8-88.8%) of infecting another household member compared to those reporting symptoms, accounting for 14.5% (95%CrI, 7.2-22.7%) of all household infections

Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed beta-propeller domain of the HIV-1 cellular interactor EED.

International audienceBACKGROUND: The human EED protein, a member of the superfamily of Polycomb group proteins, is involved in multiple cellular protein complexes. Its C-terminal domain, which is common to the four EED isoforms, contains seven repeats of a canonical WD-40 motif. EED is an interactor of three HIV-1 proteins, matrix (MA), integrase (IN) and Nef. An antiviral activity has been found to be associated with isoforms EED3 and EED4 at the late stage of HIV-1 replication, due to a negative effect on virus assembly and genomic RNA packaging. The aim of the present study was to determine the regions of the EED C-terminal core domain which were accessible and available to protein interactions, using three-dimensional (3D) protein homology modelling with a WD-40 protein of known structure, and epitope mapping of anti-EED antibodies. RESULTS: Our data suggested that the C-terminal domain of EED was folded as a seven-bladed beta-propeller protein. During the completion of our work, crystallographic data of EED became available from co-crystals of the EED C-terminal core with the N-terminal domain of its cellular partner EZH2. Our 3D-model was in good congruence with the refined structural model determined from crystallographic data, except for a unique alpha-helix in the fourth beta-blade. More importantly, the position of flexible loops and accessible beta-strands on the beta-propeller was consistent with our mapping of immunogenic epitopes and sites of interaction with HIV-1 MA and IN. Certain immunoreactive regions were found to overlap with the EZH2, MA and IN binding sites, confirming their accessibility and reactivity at the surface of EED. Crystal structure of EED showed that the two discrete regions of interaction with MA and IN did not overlap with each other, nor with the EZH2 binding pocket, but were contiguous, and formed a continuous binding groove running along the lateral face of the beta-propeller. CONCLUSION: Identification of antibody-, MA-, IN- and EZH2-binding sites at the surface of the EED isoform 3 provided a global picture of the immunogenic and protein-protein interacting regions in the EED C-terminal domain, organized as a seven-bladed beta-propeller protein. Mapping of the HIV-1 MA and IN binding sites on the 3D-model of EED core predicted that EED-bound MA and IN ligands would be in close vicinity at the surface of the beta-propeller, and that the occurrence of a ternary complex MA-EED-IN would be possible.BACKGROUND: The human EED protein, a member of the superfamily of Polycomb group proteins, is involved in multiple cellular protein complexes. Its C-terminal domain, which is common to the four EED isoforms, contains seven repeats of a canonical WD-40 motif. EED is an interactor of three HIV-1 proteins, matrix (MA), integrase (IN) and Nef. An antiviral activity has been found to be associated with isoforms EED3 and EED4 at the late stage of HIV-1 replication, due to a negative effect on virus assembly and genomic RNA packaging. The aim of the present study was to determine the regions of the EED C-terminal core domain which were accessible and available to protein interactions, using three-dimensional (3D) protein homology modelling with a WD-40 protein of known structure, and epitope mapping of anti-EED antibodies. RESULTS: Our data suggested that the C-terminal domain of EED was folded as a seven-bladed beta-propeller protein. During the completion of our work, crystallographic data of EED became available from co-crystals of the EED C-terminal core with the N-terminal domain of its cellular partner EZH2. Our 3D-model was in good congruence with the refined structural model determined from crystallographic data, except for a unique alpha-helix in the fourth beta-blade. More importantly, the position of flexible loops and accessible beta-strands on the beta-propeller was consistent with our mapping of immunogenic epitopes and sites of interaction with HIV-1 MA and IN. Certain immunoreactive regions were found to overlap with the EZH2, MA and IN binding sites, confirming their accessibility and reactivity at the surface of EED. Crystal structure of EED showed that the two discrete regions of interaction with MA and IN did not overlap with each other, nor with the EZH2 binding pocket, but were contiguous, and formed a continuous binding groove running along the lateral face of the beta-propeller. CONCLUSION: Identification of antibody-, MA-, IN- and EZH2-binding sites at the surface of the EED isoform 3 provided a global picture of the immunogenic and protein-protein interacting regions in the EED C-terminal domain, organized as a seven-bladed beta-propeller protein. Mapping of the HIV-1 MA and IN binding sites on the 3D-model of EED core predicted that EED-bound MA and IN ligands would be in close vicinity at the surface of the beta-propeller, and that the occurrence of a ternary complex MA-EED-IN would be possible

Expression, purification, crystallization and preliminary X-ray crystallographic studies of a psychrophilic cellulase from Pseudoalteromonas haloplanktis

peer reviewedThe Antarctic psychrophile Pseudoalteromonas haloplanktis produces a cold-active cellulase. To date, a three-dimensional structure of a psychrophilic cellulase has been lacking. Crystallographic studies of this cold-adapted enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of the cold adaptation and the high catalytic efficiency of the enzyme at low and moderate temperatures. The catalytic core domain of the psychrophilic cellulase CelG from P. haloplanktis has been expressed, purified and crystallized and a complete diffraction data set to 1.8 Angstrom has been collected. The space group was found to be P2(1)2(1)2(1), with unit-cell parameters a = 135.1, b = 78.4, c = 44.1 Angstrom. A molecular-replacement solution, using the structure of the mesophilic counterpart Cel5A from Erwinia chrysanthemi as a search model, has been found

Pharmacological Modulation of Platelet-derived Growth Factor Signalling with Imatinib Mesylate in a Murine Mod

Objective : Evidence for the importance of platelet-derived growth factor (PDGF) signalling in the fibrotic process is provided by 1) the fact that PDGF elicits direct fibrogenic action stimulating production of ECM 2) reports showing that a number of fibrogenic mediators such as TGFb, IL-1,TNF-α, bFGF and thrombin exhibit PDGF-dependent profibrotic activities. It has been suggested that the PDGF-PDGFR system might be a promising target for treating fibrotic diseases, thus we aimed at investigating whether the use of a tyrosin kinase inhibitor such as Imatinib mesylate (Glivec®) could reverse pulmonary fibrosis. Materials and methods: Lung fibrosis was induced in 15 pathogen-free 10-wk-old female C57/BL6 mice by IP injection of Bleomycin at 40 mg/kg body weight on Days 0, 2, 4, 6 and 8 and treated the mice with Glivec for 2 weeks with subcutaneous micro-osmotic pumps and sacrificed the 30th day. Mice were divided into 2 groups: controls treated with bleomycin alone and mice treated with bleomycin and Glivec at 50 mg/Kg. Mice follow-up was performed twice a week for the overall experimental period with check of weight. Tissue collection was performed at day 30. Fibrosis development was monitored by Hematoxilin Eosin Saffranin staining. Evolution of the molecular markers of fibrosis (CTGF, α-sm Actin, TGFβ) will be monitored by WB and Q-RT PCR. Results: In control mice treated with bleomycin alone, histopathological examination showed development of widespread fibrotic lesions with disruption of lung structure and accumulation of ECM in the sub-pleural areas. In mice treated with Glivec, lung fibrosis decreased. Inflammatory lesions persisted whereas fibrotic lesions became discontinuous and less dense. Conclusion: The present results showed that bleomycin-induced lung fibrosis can be improved by Glivec treatment. Experiments to quantify the reduction and the mechanism of reversion of fibrosis at the molecular level are currently ongoing. The next step it will be to combine Glivec with other anti-fibrotic agent (such as antioxidants or statins) to enhance the anti-fibrotic action