Biomedical Engineering Reference
In-Depth Information
Chapter 30
Application of the Proximity-Dependent Assay
and Fluorescence Imaging Approaches to Study
Viral Entry Pathways
Alex Lipovsky , Wei Zhang , Akiko Iwasaki , and Daniel DiMaio
Abstract
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 trans-
port 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 specifi c 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 fi ndings, confi rmation 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 fi ndings from a genome-wide loss-of-function genetic screen. In addition, we discuss a number of
important considerations for the utilization of immunofl uorescence microscopy and RNA interference to
investigate the molecular mechanisms of virus entry.
Key words
siRNA, Proximity ligation assay, Immunofl uorescence, Virus entry, HPV, RNA
interference
1
Introduction
High-throughput genetic screens have transformed our ability to
identify the cellular factors required for all aspects of virus infec-
tion, including entry into cells, virus replication, and virus release
from the infected cell. Loss-of-function screens based on RNA
interference have found widespread use among researchers study-
ing entry mechanisms by clinically relevant viral pathogens such as
the human immunodefi ciency virus (HIV), hepatitis C virus
