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 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:
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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Fantastic Fluorescence:Brainbow and The Nobel Prize 2008

In my childhood, I used to be fascinated by the mysterious glow of fireflies. Later I learned that it was due to a reaction between a substance called Luciferin and an enzyme, luciferase, a phenomenon called bioluminescence. This kind of glow is not limited to land creatures. Creatures living at the bottom of oceans too emit light.

Osamu Shimomura of Japan was given the task of isolating the substance which let the marine mollusk Cipridina glow when it was crushed and mixed with water. He succeeded, and on the wings of his publication, was recruited by the Princeton University, in the United States. There he began studying Aequorea Victoria, a marine jellyfish which glowed green when agitated. The jellyfish had an umbrella like shape and its outer rim glowed green. He chopped off the outer edges, crushed it, and filtered it to obtain a ‘squeezate’. He noticed one day that the ‘squeezate’ glowed blue when he poured some into the sink. He understood that it was the calcium ions (Ca++) in the seawater present in the sink that made it glow blue. It was christened aequorin.

During the extraction process they also chanced upon another protein called GFP, for green fluorescent protein. It glowed green when excited with ultraviolet light or light in the blue spectrum. The structure of GFP is barrel shaped (also likened to beer can shape), and consists of 238 amino acids. The chromophore or light emitting part is in the interior of the ‘beer can shaped’ molecule, in a region called the alpha helix region (of the molecule); while the exterior of the molecule was comprised of beta pleated sheets of the GFP molecule. Shimomura and colleagues showed that the blue color emitted by aequorin donor excited the GFP acceptor in an energy transfer process. The photons in the blue wavelength were absorbed by the GFP chromophore and photons of green wavelength were emitted - a phenomena called (bio)fluorescence. Fluorescence differs from luminescence from the fact that in fluorescence light of another wavelength is emitted than the one absorbed and luminescence means emission of light.

The whole story struck a chord in Martin Chalfie’s ears. He thought what if he could harness the gene that codes for GFP and bind it to the segment that coded for a protein of interest? He worked with a roundworm, Caenorhabditis elegans, a simple organism with only 959 cells and yet a complete organism for it could procreate, had a brain and even one third of its genes were related to humans. It was translucent and hence studying its interior was easier. He contacted Douglas Prasher who was also hot in the trail for the GFP genes. Douglas Prasher did as he promised. He sent the GFP gene to Chalfie once he got hold of the gene. Chalfie introduced it behind the promoter of the gene that coded for proteins in C elegans’ touch receptor neurons. The neurons were cleanly delineated, that too in a live worm and ‘real-time’! GFP, being a natural gene product, is non toxic.

Roger Tsien wanted more. He knew from earlier studies that the ‘chromophore’ had 3 key amino acids: serine, tyrosine and glycine in position 65, 66 and 67 respectively in the 238 amino acid long GFP molecule which formed the chromophore. He used DNA technology to alter the amino acid sequence so as to obtain GFP variants that would absorb and emit light in different part of the electromagnetic spectrum. This way he obtained cyan, yellow and blue. He obtained DsRED, a red GFP-like protein extracted from coral, from two Russian researchers and modified it so that it was stable and of desired molecular weight.

It was all set by now. Researchers now modified mice genetically and introduced the gene for red, cyan and yellow GFP. They expressed the corresponding proteins in their brain and what we got is a riot of colors, the ‘brainbow’, short for brain and rainbow. Like the elementary colors, these colors when combined in different proportions, produce many colors, just as a color printer does using them (cyan, yellow and red). One could now visualize the neural circuitry in much the same way as seeing electronic circuits. Disease detection and progression in Alzheimer’s disease, cancer and Parkinson’s disease are some potential clinical applications. Watching biogenesis of HIV1 (the virus that causes AIDS) in live cells in real time is now an easy meat. GFP can also be engineered to recognize heavy metals like cadmium (a cancer causing chemical), explosives like TNT and Arsenic (a water pollutant causing Arsenicosis).

Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry, 2008. Sadly, Doug Prasher was left out, despite his outstanding contribution in this field. He is now driving a van at $10 an hour to meet his living expenses. He is not alone. This year’s Nobel in Physiology or Medicine too left out Robert Gallo, an HIV pioneer. So, not totally a happy ending.

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References:

The Nobel Prize in Chemistry 2008
The Man Who Missed the Nobel Prize
Stuart Cantrill (2008). Nobel Prize 2008: Green fluorescent protein Nature Chemistry DOI: 10.1038/nchem.75