Super-resolution microscopy helps quantify viral DNA
In hopes of understanding more on how viruses infect cells, scientists at the University of Zurich in Switzerland turned to super-resolution microscopy to help quantify viral DNA (vDNA) trafficking.
In hopes of understanding more on how viruses infect cells, scientists at the University of Zurich in Switzerland turned to super-resolution microscopy to help quantify viral DNA (vDNA) trafficking. The method, which labels newly synthesized virus without altering infectivity, allows the visualization of infection at single-molecule resolution.
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In one of the first investigations made possible by the new method, adenovirus infection was analyzed, and it was observed that a large pool of capsid-free vDNA accumulated in the cytosol upon virus uncoating. A possible explanation is that nuclear import of incoming vDNA is a bottleneck.
Such observations suggest the usefulness of the new method, which could be applied to the entire replication cycle of DNA viruses. According to the scientists who developed the method, it offers opportunities to localize cellular and viral effector machineries on newly replicated vDNA, or innate immune sensors on cytoplasmic viral DNA.
In their paper, the scientists describe how they used cell cultures and human adenoviruses causing respiratory diseases and conjunctivitis, herpes viruses, and vaccinia virus. To label the DNA of an intact virus, the scientists turned to click chemistryâthe metabolic labeling of DNA with âclickableâ nucleoside analogs and copper(I)-catalyzed azide-alkyne cycloaddition reactions. This approach, unlike antibody- and oligonucleotide-based detection methods for DNA, allows DNA visualization without sample denaturation.
âOur molecule is incorporated into viral DNA without affecting the biological functions of the DNA,â says Prof. Nathan W. Luedtke, a study co-author from the Institute of Organic Chemistry at the University of Zurich. âAnd it can be used to label the DNA for fluorescence microscopy.â
After infecting human cells in culture with the labeled viruses, the scientists observed the behavior of vDNA during entry into cells. â[We saw] that not all the incoming vDNA enters the cell nucleus as originally expected, but a significant fraction remains in the cytosol, the fluids of the cytoplasm," noted Prof. Urs Greber, a cell biologist at the University of Zurich who led the work. According to the scientists, this phenomenon may be part of the antiviral defense reaction.
The scientists also observed that cells of the same type take up different amounts of viral DNA into their nucleus. These differences, Prof. Greber suspects, may be due to antiviral defense reactions of the nucleus, akin to those in the cytosol. Moreover, these defense reactions may vary between cells.
To further explore such questions, the new method may prove invaluable. The scientists say that their method could be applied to other DNA viruses or the HI virus (HIV).
Full results of the work appear in the journal Cell Host & Microbe; for more information, please visit http://www.cell.com/cell-host-microbe/retrieve/pii/S1931312813003247.
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