Download Case Study - Virology 4D quantitative analyses of virus

Case Study - Virology
4D quantitative analyses of virus infection
Amanda Howard and Bernard Moss, National Institute of Allergy and Infectious Disease.
Researchers from the National Institute of Allergy and Infectious Disease (NIAID) used Imaris to better understand some of the
processes that take place when an orthopoxvirus infects a cell. Orthpoxviruses include the viruses that cause smallpox, cowpox,
monkeypox and mousepox.
“The ability to surface render specific structures and easily extract
statistical data from multiple channels is a powerful feature of Imaris
that was instrumental to our study,” said Dr. Amanda Howard, who was a
postdoc in Dr. Bernard Moss’s lab at the NIAID.
When some orthopoxviruses infect cells the mature virus particles, or
virions, become embedded within large cytoplasmic matrices called
A-type inclusions (ATIs). The inclusion matrix is made of the protein ATIp,
which is thought to protect the infectivity of virions. The researchers
wanted to know how the virions become embedded in the ATIs.
“We found that microtubules are important for the trafficking of individual
virions to the ATIs and for coalescence of small ATIs into larger ones,” said
Dr. Bernard Moss, Chief of the Laboratory of Viral Diseases at the NIAID,
which is part of the National Institutes of Health.
For their experiments, the researchers used mCherry fluorescent protein
to label ATIp and yellow fluorescent protein (YFP) to label the viral core
protein in live cells. Imaris let them reconstruct 3D images from confocal
z-sections of the cells. They viewed images that showed both the mCherry
and YFP fluorescence channels in Imaris Surpass mode to quickly identify
ATIs that contained virions associated at the surface as well as those with
virions embedded within, Imaris Slice mode also helped them distinguish
between ATIs embedded throughout with virions from those that contained
virions only at the surface.
Image courtesy of Bernard Moss, NIAID.
“The ease of toggling between the two image views is a very useful
feature of the software,” Dr. Howard said.
The researchers also used Surfaces (one of the four object tools in Imaris)
to quantitatively study the effects of ATI coalescence on the accumulation
of virions. They generated surface renderings of non-coalesced and
coalesced ATIs and then extracted data from the ATIp (mCherry) and the
YFP (virions) channels within the defined ATI using Imaris. Comparing
volume data from the ATI channel with the accumulation of YFP
fluorescence showed that coalescence embeds virions within the ATI
matrix.
Finally, they performed tracking with Surfaces and Spots (two of the four
object tools available in Imaris) to quantitatively monitor the movement
of individual virions to ATIs. Imaris revealed that mature virions move
to associate with the surface of ATIs at speeds consistent with those
observed for mature virions moving along microtubules to sites of
wrapping.
Overall, the image analyses showed that microtubules have an important
role in embedding mature virions in the ATI matrix, ATI coalescence,
and the transport of mature virions to ATIs. The researchers now want
to determine how the ATI matrix protein is delivered from where it is
synthesized to the inclusion bodies. “There are some clues that the mRNA
is transported and translated in situ,” said Dr. Moss.
Time lapse video of ATI coalescence. Infected cells were imaged as Z-stacks at
20-minute intervals for 4 hours. Imaris Surfaces was used to track an individual
ATI (dragontail) and measure virion embedment as fluorescence within the defined
surface over time. ATIs=red; virions=green.
Research Paper: Howard AR, Moss B. 2012. Formation of orthopoxvirus cytoplasmic A-type inclusion bodies and embedding of virions are dynamic processes requiring microtubules. J Virol.
2012 May;86(10):5905-14.
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Case Study - Virology
4D quantitative analyses of virus infection
Amanda Howard and Bernard Moss, National Institute of Allergy and Infectious Disease.
7 ATIs are visible (red volume rendered objects). The ATI in the center of the image
was tracked (red track) as were the Virions included within it (green small spheres
indicate the location of the Virions on the last time point; gray arrows illustrate the
shortest path between start and end of each Viron track).
Close up of tracked ATI and Virions.
2D “Imaris Vantage Plot”. “Time” on the x-axis and number of Virions per ATI on the
y-axis. This plot shows the same ATI over time. The ATI is color coded based on the
number of Virions it includes.
3D “Imaris Vantage Plot”. “Time” on the x-axis, number of Virions per ATI on the
y-axis and ATI volume on the z-axis. This plot shows the same ATI over time as well
as all the Virons included. The ATI is color coded based on the number of Virions
it includes. The lines shown represent the tracks for each Virion (tracks are color
coded for time point in experiment).
Research Paper: Howard AR, Moss B. 2012. Formation of orthopoxvirus cytoplasmic A-type inclusion bodies and embedding of virions are dynamic processes requiring microtubules. J Virol.
2012 May;86(10):5905-14.
www.bitplane.com