ABSTRACT
Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.
Subject(s)
RNA Interference , Single-Cell Analysis/methods , Virus Diseases/genetics , Bayes Theorem , Cellular Microenvironment , Computer Simulation , Genomics/methods , HeLa Cells , Humans , Image Processing, Computer-Assisted/methods , Models, Biological , RNA, Small Interfering , RNA, Viral/isolation & purification , Reproducibility of Results , Systems Biology/methods , Viral Proteins/genetics , Viral Proteins/isolation & purification , Virus Diseases/metabolism , Viruses/isolation & purification , Viruses/pathogenicityABSTRACT
The formation of enveloped virus particles requires that key structural components of the virus, and the viral genomic RNA, are brought together at a cellular membrane system where new particles are assembled. The trafficking events, and the subsequent assembly and release of infectious virus particles, is co-coordinated through interactions between the viral structural proteins and cellular proteins. In the present paper, we consider how these events occur during HIV production in macrophages. In these cells, virus assembly appears to occur on a pre-existing specialized plasma membrane domain that is sequestered within the cells. The events that take place at these intracellular assembly sites may endow the virus with unique biochemical characteristics and allow virus release to be co-ordinated through the formation of infectious synapses.
