Application of the proximity-dependent assay and fluorescence imaging approaches to study viral entry pathways

Virus entry into cells is a complex, multistep process that requires the coordinated activities of a large number of cellular factors and multiple membrane compartments. Because viruses can enter cells via one or more of a large number of preexisting pathways, understanding the mechanism of virus entry and transport between various intracellular compartments is a challenging task. The arrival of "omics" technologies such as genome-wide RNA interference screens has greatly advanced our ability to study the molecular intricacies of viral entry. Bioinformatics analyses of high-throughput screen data can identify enriched gene categories and specific individual genes required for infection, which can yield important insights into the cellular compartments that viruses traverse during infection. Although there are a variety of well-established genetic and biochemical approaches to validate genome-wide screen findings, confirmation of phenotypes obtained from RNA interference studies remains an important challenge. Imaging techniques commonly used to visualize virus localization to cellular organelles are often prone to artifacts that result from the necessity of using a high multiplicity of infection. Fortunately, recent advances in microscopy-based methods for studying protein location have improved our ability to accurately pinpoint virus localization within its host cell. Here we describe in detail one such technique-the proximity ligation assay (PLA)-as a tool to validate findings from a genome-wide loss-of-function genetic screen. In addition, we discuss a number of important considerations for the utilization of immunofluorescence microscopy and RNA interference to investigate the molecular mechanisms of virus entry.