Human adenovirus (HAdV) includes a
broad group of DNA viruses that can cause infected patients to develop symptoms
ranging from respiratory infections or conjunctivitis1. Over eighty
strains of this extremely prevalent nuclear virus have been identified2.
While this virus is quite prevalent, current anti-viral medications prescribed
to people infected with the disease have only limited effectiveness against the
disease. Improved anti-viral drug treatments for HAdV are needed because this
virus can be fatal in immunocompromised hosts such as patients with AIDS, the
elderly or infants3.
One avenue for
developing an effective treatment for human adenovirus involves the
identification of small molecules that may disrupt the organization pathways
that HAdV uses to infect a cell. When infecting a human host, HAdV must rely on
proteins and molecular pathways native to the host cell to generate more
viruses that are capable of infecting more host cells. The HAdV hijacks host
cell mechanism to multiply and infect other cells in the human host. Small
molecules such as selective estrogen receptor modulators have previously been
shown to successfully disrupt the propagation of viruses like Ebola by
interfering with the viruses’ ability to manipulate host cell proteins and
pathways4. Until recently, however, very few studies have
investigated the effectiveness of small molecules in disrupting the progression
of HAdV, and those studies that have investigated the impact of small molecules
on the disruption of HAdV have failed to perform a large enough exploration of
small molecules5. Thus, the Parks laboratory at the Ottawa Hospital designed
a new experimental method for visualizing the interaction between HAdV and
small molecules that has broad application for testing purposes.
Researchers at
the Parks laboratory ultimately developed a fluoresce tagging protocol that
fused a red fluorescent protein (RFP) with the HAdV’s major late promoter
(MLP). The MLP is active during the end of HAdV replication process and,
consequently, is expressed in greater quantities when the virus is about to
spread to other cells. Greater expression of MLP in a cell indicates that the
virus is forming more viral particles as the MLP is involved in the construction
of structural proteins used for the new viruses to exit the host cell. By
fusing a RFP with the MLP, the researchers could determine whether or not
specific small molecules were able to disrupt HAdV by visualizing a reduction
in MLP activation. HAdV’s that are unable to express MLP would be unable to
reproduce effectively and thus the small molecule will have affectively disrupt
the virus.
After
manipulating the HAdV’s genome to include RFP, the virus was incubated with a
human lung-derived cell line that was tagged with a green fluorescent protein
and infection was allowed to occur. The two different fluorescent colors enabled
the researchers to optimize their study and identification technique. The
relative amount of green fluorescence permitted the researches to assess
whether or not the cells utilized in the experiment were damaged. If the cells
were damaged, a decrease in red fluoresce could be the result of a decrease in
viable cells instead of a suppression of HAdV replication. Thus, the multiple
fluorescent tags improved the validity of the Parks’ study. Researchers
quantified the level of RFP in the virally infected cell line after twenty-four
hours to determine the base level of RFP in HAdV infected human lung cell
lines.
Figure
1: Imagining
of Human Lung Cells Infected with RFP Tagged HAdV. Live-cell images of human lung cells following 18,
21, or 24 respective hours following infection with RFP tagged HAdV. Images
display the effectiveness of the tag to increase fluorescence following longer
incubation periods in cells and thus higher presence of the virus.
After testing
the validity of their viral construct through imaging seen in Figure 1, the
researchers then used their newly developed fluorescent method to examine the
effectiveness of 1200 FDA approved small molecules to inhibit the spread of
HAdV. From their screen assays, the Parks laboratory determined that digoxin,
digitoxigenin, and lanatoside C were the drugs that yielded the greatest
reduction in RFP. All three drugs are cardiotonic steroids that inhibit the
activity of Na+/K+ ATPases which are critical for the maintenance of cell
membrane potential6. Through plaque assays that measure virally
induced cell death, the experimenters also showed that exposure to dioxin,
digitoxigenin, and lanatoside C lead to a significant reduction in HAdV
pathogenicity after twenty-four hours of exposure. Cardiotonic steroids were
previously shown in another study to effectively repress the infectious
capabilities of HAdV7. The combination of the experimenters’ RFP and
viral plaque assay findings and the previous work of Grosso et al. provides
substantial support for the experimenters’ conclusion that cardiotonic steroids
have the potential to be used as effective anti-viral drugs against HAdV.
Figure
2: Impact of Cardiotonic Steroids on Expression of MLP in HAdV Infected Cells. As the concentration of
the cardiotonic steroids (dioxin, digitoxigenin, and lanatoside C) increases in
the cell culture there is a decrease in the intensity of RFP in the culture.
The decrease in RFP is far greater in the cardiotonic steroid molecules than in
the control small molecule (SAHA).
Ultimately,
the Parks’ study is important to the field of virology and the general public
for two key reasons. First, the research group was able to construct and
execute a new fluorescent technique that could be used to determine the impact
of small molecules like cardiotonic steroids on the infectiousness of HAdV. The
development of valid visualization methods to monitor HAdV is critically
important for the development of effective anti-virals. The Parks’ fluorescence
method could potentially be used to monitor the impact of other anti-HAdV
treatments beyond small molecules to inhibit the production of late viral
proteins in infect cells and thus inhibit the spread of the disease.
Additionally, the research group was able to add to the body of literature
which has suggested the cardiotonic steroid molecules could be used as an
effective anti-viral therapy against HAdV. More research on the impact of cardiotonic
steroid molecules on cell cultures infected with HAdV should be performed to
determine whether or not these molecules could be used as an effective
treatment for people infected with HAdV. Developing an effective anti-viral to
HAdV could significantly improve the survival chances and quality of life of
immunocompromised people with HAdV.
1 Lion T. (2014). Adenovirus infections in immunocompetent and
immunocompromised patients. Clinical microbiology reviews, 27(3),
441–462. doi:10.1128/CMR.00116-13
2 Binder, A. M., Biggs, H. M., Haynes, A.
K., Chommanard, C., Lu, X., Erdman, D. D., … Gerber, S. I. (2017). Human
Adenovirus Surveillance - United States, 2003-2016. MMWR. Morbidity and
mortality weekly report, 66(39), 1039–1042.
doi:10.15585/mmwr.mm6639a2
3 Bhatti, Z., & Dhamoon, A. (2017). Fatal adenovirus
infection in an immunocompetent host. The American journal of emergency
medicine, 35(7), 1034-e1.
4 Johansen, L. M., Brannan, J. M., Delos,
S. E., Shoemaker, C. J., Stossel, A., Lear, C., ... & Lehár, J. (2013).
FDA-approved selective estrogen receptor modulators inhibit Ebola virus
infection. Science translational medicine, 5(190),
190ra79-190ra79.
5 Duffy, M. R., Parker, A. L., Kalkman, E.
R., White, K., Kovalskyy, D., Kelly, S. M., & Baker, A. H. (2013).
Identification of novel small molecule inhibitors of adenovirus gene transfer
using a high throughput screening approach. Journal of controlled
release, 170(1), 132-140.
6 Prassas, I., & Diamandis, E. P.
(2008). Novel therapeutic applications of cardiac glycosides. Nature
reviews Drug discovery, 7(11), 926.
7 Grosso, F., Stoilov, P.,
Lingwood, C., Brown, M., & Cochrane, A. (2017). Suppression of adenovirus
replication by cardiotonic steroids. Journal of virology, 91(3),
e01623-16.
