RESUMEN
RNA sequencing (RNA-seq) analysis of virus-infected host cells enables researchers to study a wide range of phenomena involving host-virus interactions. This includes genomic analysis of the viral population itself, as well as analysis of the transcriptional dynamics of the virus and host during infection. In this chapter, we provide a guide for researchers interested in performing RNA-seq data analysis of virus-infected host cells or cell lines. We outline several bioinformatic protocols for quantifying viral abundance, assembling viral genomes from mixed samples, and performing differential expression analysis, among other common workflows. These workflows can be used as starting points for researchers aiming to analyze RNA-seq datasets of mixed samples containing both host and viral RNA, such as virus-infected cell lines or clinical samples.
Asunto(s)
Biología Computacional , RNA-Seq , Humanos , RNA-Seq/métodos , Biología Computacional/métodos , ARN Viral/genética , Interacciones Huésped-Patógeno/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Transcriptoma , Genoma Viral , Programas Informáticos , Virus/genética , Virosis/virología , Virosis/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Línea CelularRESUMEN
Daptomycin is a lipopeptide antibiotic that binds and permeabilizes the cell membranes of Gram-positive bacteria. Membrane permeabilization requires both calcium and phosphatidylglycerol (PG) in the target membrane, and it correlates with the formation of an oligomer that likely comprises eight subunits, which are evenly distributed between the two membrane leaflets. In both bacterial cells and model membranes, changes in the fatty acyl composition of the membrane phospholipids can prevent permeabilization. We here used liposomes to study the effect of phospholipids containing oleoyl and other fatty acyl residues on daptomycin activity, and made the following observations: (1) Oleic acid residues inhibited permeabilization when part not only of PG, but also of other phospholipids (PC or cardiolipin). (2) When included in an otherwise daptomycin-susceptible lipid mixture, even 10% of dioleoyl lipid (DOPC) can strongly inhibit permeabilization. (3) The inhibitory effect of fatty acyl residues appears to correlate more with their chain length than with unsaturation. (4) Under all conditions tested, permeabilization coincided with octamer formation, whereas tetramers were observed on membranes that were not permeabilized. Overall, our findings further support the notion that the octamer is indeed the functional transmembrane pore, and that fatty acyl residues may prevent pore formation by preventing the alignment of tetramers across the two membrane leaflets.
