January 11, 2009

Visualizing Viral Kinetics Using Fluorescence and Bioluminescence

It would be nice if we could see an individual virus particle, a virion, in real time within a mammalian tissue starting from its attachment to the host cell and entry, to its assembly and budding and release. The dynamics of viral production has been studied using computational models by noting the response of the virus to exogenous administration of reverse transcriptase and protease inhibitors. It was noted that a mind boggling 10^10 to 10^11 virions are produced each day by using this mathematical model. Now, Jouvenet et al have been able to fluorescently label a molecule called Gag protein (for group specific antigen), the major structural component of HIV. With the aid of fluorescence resonance energy transfer (FRET) and other techniques on these fluorescently tagged virions in living cells, they have been able to see the biogenesis of HIV virions in real time; from viral assembly to release by budding. The assembly rate accelerated as the Gag protein accumulated inside the cells. Typically, the time required for the assembly was just 5-6 minutes.

In fluorescence resonance energy transfer (FRET), an external light source shines on Schematic diagram of fluorescent resonant energy transfer, FRET a donor fluorescent molecule. The donor molecule gets excited and emits light of a different frequency (fluorescence), which activate the acceptor molecule. The acceptor fluorophore then emits a photon of yet another wavelength (or a quantum, as we are referring to the particle nature of light here). Both donor and acceptor fluorophores are nothing but color variants of green fluorescent protein or GFP. The whole process (FRET) is noisy as the incident light messes up with the emitted light. The incident light may also activate the acceptor fluorophore directly, leading to error.

Recently, Asokan et al used bioluminescence from Gaussia luciferase to study adeno associated virus (AAV) kinetics in living mammalian cells. By using bioluminescent molecules, the external light source as used in FRET was no longer needed. This way, direct activation of acceptor molecule was avoided and background noise was kept to a minimum. They first amplified gLuc (Gaussia luciferase) in a plasmid by polymerase chain reaction or PCR, using primer sequences. They then fused the resulting protein to that of an adenoviral subunit of AAV, called Vp2. The resulting gLuc/AAV construct was then injected into the left hind limb of rats. They could clearly notice the AAV vector dynamics. The importance of such dynamics is realized when the use of AAV as a vector in gene therapy is considered. They opined that such a technique would be ideal in studying viral dynamics in peripheral tissues such as the eye and the brain.

Bioluminescence is used to study virus tropism and viral kinetics. Tropism refers to the different populations of host cells a virus can attack. Retroviruses have a narrow tropism meaning they can infect only a few types of cells such as CD4+ T cells and macrophages. Previous studies employed Gaussia luciferase reporter gene as a tool for studying viral dynamics. Recent experiments promise a better future for the study of viral behavior.

References:
ResearchBlogging.org A Asokan, J S Johnson, C Li, R J Samulski (2008). Bioluminescent virion shells: new tools for quantitation of AAV vector dynamics in cells and live animals Gene Therapy, 15 (24), 1618-1622 DOI: 10.1038/gt.2008.127

Human Immunodeficiency Virus Disease: AIDS and Related Disorders: Anthony S. Fauci, H. Clifford Lane, Harrison’s Principles of Internal Medicine, 17th Ed.

Imaging the biogenesis of individual HIV-1 virions in live cells
Nolwenn Jouvenet, Paul D. Bieniasz, & Sanford M. Simon
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