The adaptation of CRISPR/Cas technology for use in mammals has revolutionized genome engineering. In particular with regard to clinical application, efficient expression of Cas9 within a narrow time frame is highly desirable to minimize the accumulation of off-target editing. We developed an effective, aptamer-independent retroviral delivery system for Cas9 mRNAs that takes advantage of a unique foamy virus (FV) capability: the efficient encapsidation and transfer of non-viral RNAs. This enabled us to create a FV vector toolbox for efficient, transient delivery (TraFo) of CRISPR/Cas9 components into different target tissues. Co-delivery of Cas9 mRNA by TraFo-Cas9 vectors in combination with retroviral, integration-deficient single guide RNA (sgRNA) expression enhanced efficacy and specificity of gene-inactivation compared with CRISPR/Cas9 lentiviral vector systems. Furthermore, separate TraFo-Cas9 delivery allowed the optional inclusion of a repair matrix for efficient gene correction or tagging as well as the addition of fluorescent negative selection markers for easy identification of off-target editing or incorrect repair events. Thus, the TraFo CRISPR toolbox represents an interesting alternative technology for gene inactivation and gene editing.
Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells.
Table of contents Oral presentations Session 1: Entry & uncoating O1 Host cell polo-like kinases (PLKs) promote early prototype foamy virus (PFV) replication Irena Zurnic, Sylvia Hütter, Ute Lehmann, Nicole Stanke, Juliane Reh, Tobias Kern, Fabian Lindel, Gesche Gerresheim, Martin Hamann, Erik Müllers, Paul Lesbats, Peter Cherepanov, Erik Serrao, Alan Engelman, Dirk Lindemann O2 A novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized HIV-1 infection Claire Da Silva Santos, Kevin Tartour, Andrea Cimarelli O3 Dynamics of nuclear envelope association and nuclear import of HIV-1 complexes Rya Burdick, Jianbo Chen, Jaya Sastri, Wei-Shau Hu, Vinay Pathak O4 Human papillomavirus protein E4 potently enhances the susceptibility to HIV infection Oliver T. Keppler Session 2: Reverse transcription & integration O5 Structure and function of HIV-1 integrase post translational modifications Karine Pradeau, Sylvia Eiler, Nicolas Levy, Sarah Lennon, Sarah Cianferani, Stéphane Emiliani, Marc Ruff O6 Regulation of retroviral integration by RNA polymerase II associated factors and chromatin structure Vincent Parissi Session 3: Transcription and latency O7 A novel single-cell analysis pipeline to identify specific biomarkers of HIV permissiveness Sylvie Rato, Antonio Rausell, Miguel Munoz, Amalio Telenti, Angela Ciuffi O8 A capsid-dependent integration program linking T cell activation to HIV-1 gene expression Alexander Zhyvoloup, Anat Melamed, Ian Anderson, Delphine Planas, Janos Kriston-Vizi, Robin Ketteler, Chen-Hsuin Lee, Andy Merritt, Petronela Ancuta, Charles Bangham, Ariberto Fassati O9 Characterisation of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome Anthony Rodari, Benoit Van Driessche, Mathilde Galais, Nadége Delacourt, Sylvain Fauquenoy, Caroline Vanhulle, Anna Kula, Arsène Burny, Olivier Rohr, Carine Van Lint O10 Tissue-specific dendritic cells differentially modulate latent HIV-1 reservoirs Thijs van Montfort, Renee van der Sluis, Dave Speijer, Ben Berkhout Session 4: RNA trafficking & packaging O11 A novel cis -acting element affecting HIV replication Bo Meng, Andrzej Rutkowski, Neil Berry, Lars Dölken, Andrew Lever O12 Tolerance of HIV’s late gene expression towards stepwise codon adaptation Thomas Schuster, Benedikt Asbach, Ralf Wagner Session 5: Assembly & release O13 Importance of the tax-inducible actin-bundling protein fascin for transmission of human T cell leukemia virus Type 1 (HTLV-1) Christine Gross, Veit Wiesmann, Martina Kalmer, Thomas Wittenberg, Jan Gettemans, Andrea K. Thoma-Kress O14 Lentiviral nef prote...
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