Biomedical Engineering Reference
In-Depth Information
1.2 Imaging
and Biochemical
Techniques to Study
Virus Traffi cking
and Disassembly
Immunofl uorescence (IF) microscopy remains one of the most
common methods for studying virus entry. It is a powerful approach
that can sometimes reveal virus within distinct cellular organelles
without the need for complex and time-consuming biochemical
analyses. When combined with live-cell imaging, IF-based imaging
studies can shed important light on the spatiotemporal dynamics
of virus internalization and traffi cking. However, because effi cient
visualization of viruses usually requires a very high multiplicity of
infection and because of the large particle-to-infectivity ratio for
most viruses, IF is often unable to differentiate between infectious
particles and those that enter nonproductive pathways. Moreover,
subjecting cells to very high doses of pathogen can sometimes lead
to nonphysiological, cytopathic effects. Therefore, although IF is a
useful tool to study virus entry, it is far from ideal. A biochemical
approach to study virus entry is immunoprecipitation (IP), which
can detect physical association between the viral structural proteins
and host cell proteins and therefore confi rm the presence of virus
within a particular subcellular compartment. And even though IP,
often coupled to protein identifi cation by mass spectrometry, is a
powerful approach to identify individual components of a multi-
subunit complex, non-interacting or many transiently associated
proteins cannot be detected by IP, even when they are in close
proximity to the virus.
1.3 Proximity
Ligation and Flow
Cytometry Assays
to Track Virus
Movement
and Disassembly
The development of the proximity-dependent DNA ligation assay
(PLA) has provided a sensitive tool to examine protein-protein inter-
actions by taking advantage of two DNA aptamers, whose hybridiza-
tion to a target probe produces an amplifi able DNA sequence [
10
].
Developed in part as an alternative to enzyme-linked immunosor-
bent assay (ELISA) for ultrasensitive detection of very small protein
quantities in clinical samples, PLA was subsequently adapted to the
study of protein-protein interactions in situ [
11
]. This was achieved
by conjugating the DNA aptamers to antibodies specifi c for the two
targets to be assayed. Hybridization of the aptamers to a common
probe, which only occurs when the two target molecules are in close
proximity, creates a template for rolling circle DNA amplifi cation
(RCA) by a polymerase chain reaction. The single-stranded RCA
product is then incubated with fl uorescent oligonucleotides and the
resulting hybrid visualized as a single microscopic fl uorescent spot.
The number and fl uorescence intensity of PLA signals within an
image can be conveniently quantitated by dedicated imaging soft-
ware. The PLA technology is especially powerful when combined
with fl ow cytometry, since it allows for rapid quantifi cation of pro-
tein-protein interactions within a large cell population [
12
].
Although in situ PLA was developed and most often applied
to the study of proteins within a putative complex, it can detect
targets that are not necessarily in direct physical contact [
13
].
