RESUMEN
The circulation of seasonal influenza A viruses (IAVs) in humans relies on effective evasion and subversion of the host immune response. While the evolution of seasonal H1N1 and H3N2 viruses to avoid humoral immunity is well characterized, relatively little is known about the evolution of innate immune antagonism phenotypes in these viruses. Numerous studies have established that only a small subset of infected cells is responsible for initiating the type I and type III interferon (IFN) response during IAV infection, emphasizing the importance of single cell studies to accurately characterize the IFN response during infection. We developed a flow cytometry-based method to examine transcriptional changes in IFN and interferon stimulated gene (ISG) expression at the single cell level. We observed that NS segments derived from seasonal H3N2 viruses are more efficient at antagonizing IFN signaling but less effective at suppressing IFN induction, compared to the pdm2009 H1N1 lineage. We compared a collection of NS segments spanning the natural history of the current seasonal IAV lineages and demonstrate long periods of stability in IFN antagonism potential, punctuated by occasional phenotypic shifts. Altogether, our data reveal significant differences in how seasonal and pandemic H1N1 and H3N2 viruses antagonize the human IFN response at the single cell level.
RESUMEN
Characterizing the molecular interactions of viruses in natural microbial populations offers insights into virus-host dynamics in complex ecosystems. We identify the resistance of Sulfolobus islandicus to Sulfolobus spindle-shaped virus (SSV9) conferred by chromosomal deletions of pilin genes, pilA1 and pilA2 that are individually able to complement resistance. Mutants with deletions of both pilA1 and pilA2 or the prepilin peptidase, PibD, show the reduction in the number of pilins observed in TEM and reduced surface adherence but still adsorb SSV9. The proteinaceous outer S-layer proteins, SlaA and SlaB, are not required for adsorption nor infection demonstrating that the S-layer is not the primary receptor for SSV9 surface binding. Strains lacking both pilins are resistant to a broad panel of SSVs as well as a panel of unrelated S. islandicus rod-shaped viruses (SIRVs). Unlike SSV9, we show that pilA1 or pilA2 is required for SIRV8 adsorption. In sequenced Sulfolobus strains from around the globe, one copy of each pilA1 and pilA2 is maintained and show codon-level diversification, demonstrating their importance in nature. By characterizing the molecular interactions at the initiation of infection between S. islandicus and two different types of viruses we hope to increase the understanding of virus-host interactions in the archaeal domain.
Asunto(s)
Resistencia a la Enfermedad/genética , Proteínas Fimbrias/metabolismo , Fuselloviridae/fisiología , Interacciones Microbiota-Huesped , Rudiviridae/fisiología , Sulfolobus , Proteínas Fimbrias/genética , Fimbrias Bacterianas/metabolismo , Sulfolobus/genética , Sulfolobus/virología , Acoplamiento ViralRESUMEN
Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.
Asunto(s)
Desoxirribonucleasas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Receptores CCR5/metabolismo , Dedos de Zinc , Animales , Proteínas de Unión al ADN/genética , Desoxirribonucleasas/genética , Genes Reporteros , Genoma , Humanos , Biblioteca de Péptidos , Receptores CCR2/metabolismoRESUMEN
Cys2His2 zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain's well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain-DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain-DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities.
Asunto(s)
Cisteína/química , ADN/metabolismo , Histidina/química , Dedos de Zinc , Sitios de Unión , ADN/química , Recolección de DatosRESUMEN
The Cys2His2 zinc finger (ZF) is the most frequently found sequence-specific DNA-binding domain in eukaryotic proteins. The ZF's modular protein-DNA interface has also served as a platform for genome engineering applications. Despite decades of intense study, a predictive understanding of the DNA-binding specificities of either natural or engineered ZF domains remains elusive. To help fill this gap, we developed an integrated experimental-computational approach to enrich and recover distinct groups of ZFs that bind common targets. To showcase the power of our approach, we built several large ZF libraries and demonstrated their excellent diversity. As proof of principle, we used one of these ZF libraries to select and recover thousands of ZFs that bind several 3-nt targets of interest. We were then able to computationally cluster these recovered ZFs to reveal several distinct classes of proteins, all recovered from a single selection, to bind the same target. Finally, for each target studied, we confirmed that one or more representative ZFs yield the desired specificity. In sum, the described approach enables comprehensive large-scale selection and characterization of ZF specificities and should be a great aid in furthering our understanding of the ZF domain.