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Observing Viruses on a Live Stream

14 Jul | By Biophotonics.World
Observing Viruses on a Live Stream
Image source: Leibniz IPHT
By: Sven Döring

Christian Eggeling places a ring of light around the focus of the samples he examines under the microscope – an optical trick to bypass the optical resolution limit described by Ernst Abbe. This enables Eggeling and his international team of researchers to observe processes deep inside living cells 

Researchers visualize
how the AIDS pathogen multiplies in the body. This helps to identify targets for new therapies 

AIDS is caused by human immuno- deficiency viruses (HIV) that put immune cells — so-called T helper cells — out of action. Instead of controlling other immune system cells in the defence against pathogens, infected T helper cells produce new HI viruses in large quantities. An international research team around Christian Eggeling has now managed to observe the spread of human immunodeficiency viruses amongst living T helper cells in real time with the help of ultra-high-resolution imaging. 

Using super-resolution STED fluorescence microscopy, the researchers provide direct proof for the first time that the AIDS pathogen creates a certain lipid environment for replication. "This gives us a few starting points when it comes to investigating how to potentially prevent this reproduction," says Christian Eggeling, who conducts research and teaches at Leibniz IPHT, Friedrich Schiller University Jena, and the University of Oxford. 

Together with a team led by Delphine Muriaux and Cyril Favard from the Université Montpellier and his colleague Jakub Chojnacki, Christian Eggeling has been examining the plasma membrane of infected T helper cells. They focused on the "gate" through which the HI virus buds from the cell after multiplying inside. The "Gag" protein, which co- ordinates the processes involved in the assembly of the newly produced virus particles, served as a marker. "Where this protein accumulates, the decisive processes take place that lead to the virus releasing itself and infecting other cells,“ explains Christian Eggeling. In order to decipher these processes, the researchers examined the diffusion of the lipid molecules to and at the place where the "Gag" proteins gather i.e. where the virus particle buds. During the budding process, the virus particles exit the cell through the plasma membrane and receive their lipid coating. Eggeling and his colleagues have now discovered that, only certain lipids from the cell membrane interact with the HI virus. Although these lipids were already known, the research team has managed to directly prove this interaction in living cells for the first time. 

Point of attack to prevent the reproduction of the virus 

"This provides us with a potential target for antiviral drugs," says Christian Eggeling. "Knowing which molecules the HI virus needs to exit the cell and multiply is a crucial prerequisite for investigating how this can be prevented. Our technology enables us to follow the developments directly in real time". Eggeling is now working with his team on the development of antibodies to attack precisely these molecules – and thus suppress the spread of the virus. 

"We not only want to examine these antibodies from a medical perspective, but also to find out how their biophysical interaction can be exploited to make them more effective," states Eggeling. He is trying to understand how diseases develop on a molecular level by combining super-resolution fluorescence microscopy with technology that tracks the movement of labeled molecules in real time. Eggeling helped develop the STED microscopy method during his time in the laboratory of Stefan Hell in Göttingen, who went on to win the Nobel Prize in chemistry in 2014. This enables the spatial and temporal examination of individual molecules in living cells. "We can now reveal cellular mechanisms on a molecular level. These mechanisms were much too fast and occurred over far too small spatial scales for previous method of investigation." 

STED Microscopy 

STED stands for "Stimulated Emission Depletion" and is a method used in fluorescence microscopy that allows the optical resolution limit described by Ernst Abbe to be bypassed. Light is used to excite, fluorescent dyes, which then spontaneous- ly emit light in a lower-energy wavelength range. This spontaneous emission can be suppressed with the addition of high-intensity light at the wavelength range of emission. The de-excitation light is placed in a ring around the focus of the sample to be examined, restricting the emission of fluorescent light to the center of the sample. This optical trick makes the effective focal point significantly smaller, and its dimensions are be- low the Abbe diffraction limit. 



Publications: C. Favard et al.: HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells cre- ating a nanodomain platform for virus assembly. Science Advances (2019), DOI: 10.1126/sciadv.aaw8651 


Source: Leibniz IPHT




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