Flavivirus

Basic information and current topics regarding Flaviviridae

Review of siRNA Profiling in Dengue Virus

Note:

All images used in this review are from the below citation and have been reprinted under the Creative Commons Attribution License.

Ang F., Wong P. Y., Ng M. M., Chu J. J. (2010). Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus. Virology journal (1743-422X), 7, p. 24.


Dengue Virus (DENV) has been one of the more troublesome flaviviruses when it comes to vaccines. Although several vaccines are under development, there have been few attempts at finding a direct treatment for infection. Most protocols involve treating the individual symptoms, rather than utilizing more advanced antiviral treatment. The article reviewed here takes the first few steps in producing effective antivirals for the treatment of DENV infection.

siRNA’s have many purposes in the regulation of cellular pathways, but they also posses a good potential for working as ‘probes’ in finding host transcription profiles. Briefly, the idea is that a short strand of complementary RNA will bind to an mRNA in the subject’s cytoplasm, thus ‘interfering’ with the translation of that mRNA. Utilizing this principle, one can then target a protein thought to have some function in the subject cell, insert the siRNA corresponding to that protein’s RNA sequence, and then assay the changes produced when the protein isn’t being made. In this study, genes thought to be responsible for DENV infection were targeted to be ‘silenced.’

Immunofluorscent microscopy is one of the better and easier methods used to visualize viruses. TEM and SEM images lack the ability to show virus activity in a single cell over time. By tagging fluor-loaded antibodies to viruses, proteins, and other components, we are able to see what would otherwise be too small for light microscopes—it’s like duct-taping a flashlight to a virus then letting it loose in the cell. In the below image, the nucleus, DENV, and clathrin—a protein which builds a shell around the early-endosome—have flashlights duct-taped to them:

Ang F., Wong P. Y., Ng M. M., Chu J. J. (2010). Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus. Virology journal (1743-422X), 7, p. 24.

By looking at the fourth column, row C, we find that DENV attachment/entry stimulates the production of clathrin. This also shows perfect little yellow dots, which suggest the DENV (tagged with green) is overlapping the location of clathrin (tagged red)—supporting the notion that clathrin plays a role in DENV entry. Below, this idea is further supported by using a mutant (left image) that is unable to produce clathrin (DENV shown in yellow):

Ang F., Wong P. Y., Ng M. M., Chu J. J. (2010). Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus. Virology journal (1743-422X), 7, p. 24.

Clathrin is, unfortunately, a rather vital protein in cells and inhibition thereof doesn’t represent the greatest way to keep DENV out of the cell—but it is effective! Shown here is the siRNA-implementation of this idea, called ‘siRNA knockdown’ of gene products.

Ang F., Wong P. Y., Ng M. M., Chu J. J. (2010). Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus. Virology journal (1743-422X), 7, p. 24.

 Another consideration for DENV antivirals comes from the pH-mediated membrane fusion of DENV and the endosome. Shown below are DENV particles moving into the cell, yellow dots are endosomes containing Dengue Viruses.

Ang F., Wong P. Y., Ng M. M., Chu J. J. (2010). Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus. Virology journal (1743-422X), 7, p. 24.

This study did a wonderful job in capturing DENV visually, but the pictures were not the main point of the article. This—very possibly the world’s longest published bar graph—was the crux of their study:

Ang F., Wong P. Y., Ng M. M., Chu J. J. (2010). Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus. Virology journal (1743-422X), 7, p. 24.

One hundred and nineteen siRNA’s, each targeting a specific gene, were reverse-transfected into a human liver cell-line (HUH7). Under their results section, they list the optimization of a siRNA ‘screening’ assay as their first topic—after testing 119 samples of DENV-infected cells multiple times, I don’t blame them. If you’re interested in the specifics behind the genes they targeted, the authors provide a great description/reasoning for chosen genes here.

In the end, a large amount of genes were tested for their involvement with DENV infection. Although killing Dengue Viruses with a shotgun is a touch inefficient, the impact is all-the-same. It seems as though a lot of the genes chosen were candidates for having their derivatives (the ones much further down any applicable pathway) as targets for future investigation, since a lot of these gene-products are vital to the vitality of the cell. Cytotoxicity assays seemed to be downplayed quite a bit, and lacks a quantitative data report. Nonetheless, they contributed and reported on 119 genes and their involvement in DENV infection, which certainly sets the stage for future antiviral investigations.


References

Ang F., Wong P. Y., Ng M. M., Chu J. J. (2010). Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus. Virology journal (1743-422X), 7, p. 24.