SUMMARY Accessory proteins of lentiviruses, such as HIV-1, target cellular restriction factors to enhance viral replication. Systematic analyses of proteins that are targeted for degradation by HIV-1 accessory proteins may provide a better understanding of viral immune evasion strategies. Here, we describe a high-throughput platform developed to study cellular protein stability in a highly parallelized matrix format. We used this approach to identify cellular targets of the HIV-1 accessory protein Vpu through arrayed coexpression with 433 interferon-stimulated genes, followed by differential fluorescent labeling and automated image analysis. Among the previously unreported Vpu targets identified by this approach, we find that the E2 ligase mediating ISG15 conjugation, UBE2L6, and the transmembrane protein PLP2 are targeted by Vpu during HIV-1 infection to facilitate late-stage replication. This study provides a framework for the systematic and high-throughput evaluation of protein stability and establishes a more comprehensive portrait of cellular Vpu targets.
Recent studies have reported that genome editing by CRISPR–Cas9 induces a DNA damage response mediated by p53 in primary cells hampering their growth. This could lead to a selection of cells with pre-existing p53 mutations. In this study, employing an integrated computational and experimental framework, we systematically investigated the possibility of selection of additional cancer driver mutations during CRISPR-Cas9 gene editing. We first confirm the previous findings of the selection for pre-existing p53 mutations by CRISPR-Cas9. We next demonstrate that similar to p53, wildtype KRAS may also hamper the growth of Cas9-edited cells, potentially conferring a selective advantage to pre-existing KRAS-mutant cells. These selective effects are widespread, extending across cell-types and methods of CRISPR-Cas9 delivery and the strength of selection depends on the sgRNA sequence and the gene being edited. The selection for pre-existing p53 or KRAS mutations may confound CRISPR-Cas9 screens in cancer cells and more importantly, calls for monitoring patients undergoing CRISPR-Cas9-based editing for clinical therapeutics for pre-existing p53 and KRAS mutations.
The multifunctional NS1 protein of influenza A viruses suppresses host cellular defense mechanisms and subverts other cellular functions. We report here on a new role for NS1 in modifying cell-cell signaling via the Hedgehog (Hh) pathway. Genetic epistasis experiments and FRET-FLIM assays in Drosophila suggest that NS1 interacts directly with the transcriptional mediator, Ci/Gli1. We further confirmed that Hh target genes are activated cell-autonomously in transfected human lung epithelial cells expressing NS1, and in infected mouse lungs. We identified a point mutation in NS1, A122V, that modulates this activity in a context-dependent fashion. When the A122V mutation was incorporated into a mouse-adapted influenza A virus, it cell-autonomously enhanced expression of some Hh targets in the mouse lung, including IL6, and hastened lethality. These results indicate that, in addition to its multiple intracellular functions, NS1 also modifies a highly conserved signaling pathway, at least in part via cell autonomous activities. We discuss how this new Hh modulating function of NS1 may influence host lethality, possibly through controlling cytokine production, and how these new insights provide potential strategies for combating infection.
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