YSM Issue 93.2
NEWSBiochemistryTHE DUALITYOF THEEBOLA VIRUSHow a Deadly Viral InfectionCan Be Harnessed for HealingBY SYDNEY HIRSCH IMAGE COURTESY OF FLICKRWhen we hear “Ebola,” we often think of its contagion andlethality, and of the outbreaks in recent years. Ironically,scientists are exploring the potential of the deadly Ebolavirus (EBOV) as a treatment against a fatal form of cancer: braintumors. Cancer cells lack the ability to generate an immune responseagainst viruses, making viruses a good starting point for developingtreatments. Of course, infecting someone with a lethal virus is risky; tocircumvent this, scientists use chimeric viruses, which contain a mix ofgenes from multiple parent viruses. A team of researchers, includingYale professor Anthony Van den Pol, recently reported their efforts totest three variations of a chimeric virus, pairing an EBOV glycoproteinwith the vesicular stomatitis virus (VSV). They chose the Ebola gene,given the virus’s propensity to infect—and for their purposes, target—nerve tissue. Specifically, they took interest in the mucin-like domain(MLD) of the Ebola virus, and how it modulates the viral ability totarget brain tumors. Interestingly, it seemed as if the MLD protectednormal cells from infection, while cancer cells still became infected.They were hopeful that VSV-EBOV could be a promising treatment,as the combination had been an effective and safe vaccine in humansduring the African Ebola outbreak.The team tested three viruses on severe combined immunodeficient(SCID) mice, which had human brain tumor cells injected into theirbrains: VSV-EBOV, which contains the mucin-like domain (MLD);VSV-EBOVΔMLD, which is a parallel construct but lacks the mucinlikedomain; and VSV-EBOVΔMLD-GFP, which is almost identicalto VSV-EBOVΔMLD with an added green-fluorescent protein (GFP)reporter gene to visualize a virus. All three showed some increase inthe mice’s survival. The researchers found that the VSV-EBOV wasmost effective in treating the brain tumors while maintaining thehealth of the mouse. At 120 days after the tumor implant, only miceinfected with the MLD-containing virus remained alive.The researchers considered VSV-EBOV successful because itminimally infected healthy neural cells while still targeting tumorcells. Van den Pol’s team quantified the extent of the brain infectionby counting the number of infected neurons and glial cells in coronalbrain sections. The other two virus forms showed widespread infectionthroughout the brains of the animals. The VSV-EBOVΔMLD-GFP wasthe least effective. While it modestly extended the survival of the mice,all of the mice died. Some were incompletely infected by the virus, andmany still had brain tumors. The VSV-EBOVΔMLD injected tumorshad similar tissue structure, and a greater survival rate.The lethality of the VSV-EBOVΔMLD-GFP virus may have beendue to the VSV backbone itself; this differs from those of the non-GFP chimeric viruses in all four of their base proteins, which mayalter the behavior of the virus. Van den Pol explained that the Ebolavirus may release the MLD as a “false leader, causing the immunesystem to be lured away from the infected cells.” This slowedreplication of the virus and lessened the amount of infectious viraloffspring. With a slower replication rate, the innate immune systemhas more time to upregulate antiviral defenses. The low number ofinfected normal cells suggested that the innate immune system wassufficient to prevent the spread of the virus.Researchers also compared the effects of intravenous versusintracranial injection. Both methods had degrees of success.Intracranial injection showed greater tumor infection and elimination,indicating this type of delivery may be more reliable for treating largertumors. Intravenous injections, which are done through the tail-veinin mice, could on the whole be more effective for smaller or undetectedtypes of metastatic cancer, such as melanomas.Van den Pol and his team were able to monitor the impact of theMLD on the treatment and survival of SCID mice. The chimericvirus containing the mucin-like domain, VSV-EBOV, was the mostsuccessful treatment, confirming their initial expectations. Thisresearch is promising, as it could open the door for new forms ofglioblastoma treatment. “VSV-EBOV has been successfully used inthe human population in the past, showing that it’s relatively safe. Ifwe’re ultimately trying to move toward clinical studies, that’s a hurdlealready jumped over,” Van den Pol said. Future directions includelooking at tumors in immunocompetent mice or exploring otherVSV-based viruses. ■Zhang, X., Zhang, T., Davis, J.N., Marzi, A., Marchese, A.M., Robek,M.D., & van den Pol, A.N. (2020). Mucin-Like Domain of EbolaVirus Glycoprotein Enhances Selective Oncolytic Actions againstBrain Tumors. Journal of Virology, 94(8), e01967-19.8 Yale Scientific Magazine September 2020 www.yalescientific.org
Molecular BiologyNEWSMICROMOLECULE,HUGE IMPACTHow microRNAs InhibitAsthmatic ReactionsIMAGE COURTESY OF RYAN JEFFSDespite its name, microRNA helps the body on a macroscale. The small molecule, only about 22 nucleotideslong, plays an important role in the regulation ofgene expression by binding to mRNA at certain points in itssequence. At the Yale School of Medicine, Shervin Takyar andhis team investigated the role microRNAs played in controllingeosinophilia through endothelial cells. Eosinophilia occurs whenlarge numbers of white blood cells called eosinophils are recruitedto a site in the body, leading to the allergic airway reactioncharacteristic of asthma and chronic rhinosinusitis (CRS).The paper’s first point of analysis was investigating whichinhibitory factors interact with VEGF (vascular endothelialgrowth factor). “We looked at a vascular factor [endothelial cells]and inhibition, investigating their role in asthma and CRS,” Takyarexplained. Takyar’s team found that microRNA-1 levels went downwhen VEGF went up. “The next step[s] [were] figuring out first,whether microRNA-1 levels are important in the disease, andsecond, why,” Takyar said. The team was then able to show, throughbiological and mathematical models, that the molecule wasimportant. They also proved that asthmatic eosinophilia reactionswere reduced by increased levels of microRNA-1 in the blood.Takyar and his team recreated the conditions first intransgenic mice and in an engineered lentivirus (retroviruseswith long incubation periods) and then created a model fora human vessel in the lab. “We isolated endothelial cells andreversed the change [in microRNA levels], and only changingmicroRNA-1 levels in these cells [could] decrease the featuresof asthma,” Takyar said.The next step was to understand where the molecule wasacting in the cell. The results revealed that microRNA wasinhibiting the asthmatic reaction by acting in a known proteincomplex, the RNA induced silencing complex. This wasespecially hard to investigate because microRNA cannot betagged, since this would inhibit the molecule from enteringthe complex. Within the complex, there is an Argonauteprotein, which acts as a “matchmaker” for microRNA andthe inhibiting molecules; this protein was used to understandhow microRNA was influencing the eosinophilic reaction.www.yalescientific.orgBY BEATRIZ HORTA“We captured the Argonaute when microRNA was enteringto see what microRNA-1 was acting on,” Takyar said. Withthis strategy, the team identified four genes that controleosinophilia, an important breakthrough.After this discovery, the researchers extracted cells fromhumans with CRS and created a model for the humanendothelium environment. In this model, they passedeosinophils over the cells to see how many would stick to themin varying levels of microRNA. As expected, they found that inincreased levels of microRNA, less eosinophils would adhereto the cells, which revealed the mechanism of action for theinhibition of the symptoms.To the researchers, the discovery shows promise for clinicaltreatments. “Right now, we are starting a collaboration withclinical groups and some companies to use [this mechanism]as a complementary treatment for some patients who do notrespond to drug treatments,” Takyar said. He explained thattreatment for CRS and asthma is difficult because patientshave different symptoms and mechanisms; therefore, manydrugs do not have any effect on certain individuals. The roleof microRNA in the eosinophilic mechanism is an excitingdevelopment in the area and could represent a step forward toimprove treatments. ■Korde, A., Ahangari, F., Haslip, M., Zhang, X., Liu, Q., Cohn, L., Gomez,J.L., Chupp, G., Pober, J.S., Gonzalez, A., Takyar, S.S. (2020). Anendothelial microRNA-1-regulated network controls eosinophilThe Journal of Allergyand Clinical Immunology, 145(2), 550–62. https://doi.org/10.1016/j.jaci.2019.10.031Jeffs, R. (2011). MicroRNA and mRNA visualization in differentiatingC1C12 cells. Retrieved 22 March 2020, from https://commons.wikimedia.org/wiki/File:MicroRNA_and_mRNA_visualization_in_differentiating_C1C12_cells.jpg Lentivirus.aspxSeptember 2020 Yale Scientific Magazine 9
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Molecular Biology
NEWS
MICRO
MOLECULE,
HUGE IMPACT
How microRNAs Inhibit
Asthmatic Reactions
IMAGE COURTESY OF RYAN JEFFS
Despite its name, microRNA helps the body on a macro
scale. The small molecule, only about 22 nucleotides
long, plays an important role in the regulation of
gene expression by binding to mRNA at certain points in its
sequence. At the Yale School of Medicine, Shervin Takyar and
his team investigated the role microRNAs played in controlling
eosinophilia through endothelial cells. Eosinophilia occurs when
large numbers of white blood cells called eosinophils are recruited
to a site in the body, leading to the allergic airway reaction
characteristic of asthma and chronic rhinosinusitis (CRS).
The paper’s first point of analysis was investigating which
inhibitory factors interact with VEGF (vascular endothelial
growth factor). “We looked at a vascular factor [endothelial cells]
and inhibition, investigating their role in asthma and CRS,” Takyar
explained. Takyar’s team found that microRNA-1 levels went down
when VEGF went up. “The next step[s] [were] figuring out first,
whether microRNA-1 levels are important in the disease, and
second, why,” Takyar said. The team was then able to show, through
biological and mathematical models, that the molecule was
important. They also proved that asthmatic eosinophilia reactions
were reduced by increased levels of microRNA-1 in the blood.
Takyar and his team recreated the conditions first in
transgenic mice and in an engineered lentivirus (retroviruses
with long incubation periods) and then created a model for
a human vessel in the lab. “We isolated endothelial cells and
reversed the change [in microRNA levels], and only changing
microRNA-1 levels in these cells [could] decrease the features
of asthma,” Takyar said.
The next step was to understand where the molecule was
acting in the cell. The results revealed that microRNA was
inhibiting the asthmatic reaction by acting in a known protein
complex, the RNA induced silencing complex. This was
especially hard to investigate because microRNA cannot be
tagged, since this would inhibit the molecule from entering
the complex. Within the complex, there is an Argonaute
protein, which acts as a “matchmaker” for microRNA and
the inhibiting molecules; this protein was used to understand
how microRNA was influencing the eosinophilic reaction.
www.yalescientific.org
BY BEATRIZ HORTA
“We captured the Argonaute when microRNA was entering
to see what microRNA-1 was acting on,” Takyar said. With
this strategy, the team identified four genes that control
eosinophilia, an important breakthrough.
After this discovery, the researchers extracted cells from
humans with CRS and created a model for the human
endothelium environment. In this model, they passed
eosinophils over the cells to see how many would stick to them
in varying levels of microRNA. As expected, they found that in
increased levels of microRNA, less eosinophils would adhere
to the cells, which revealed the mechanism of action for the
inhibition of the symptoms.
To the researchers, the discovery shows promise for clinical
treatments. “Right now, we are starting a collaboration with
clinical groups and some companies to use [this mechanism]
as a complementary treatment for some patients who do not
respond to drug treatments,” Takyar said. He explained that
treatment for CRS and asthma is difficult because patients
have different symptoms and mechanisms; therefore, many
drugs do not have any effect on certain individuals. The role
of microRNA in the eosinophilic mechanism is an exciting
development in the area and could represent a step forward to
improve treatments. ■
Korde, A., Ahangari, F., Haslip, M., Zhang, X., Liu, Q., Cohn, L., Gomez,
J.L., Chupp, G., Pober, J.S., Gonzalez, A., Takyar, S.S. (2020). An
endothelial microRNA-1-regulated network controls eosinophil
The Journal of Allergy
and Clinical Immunology, 145(2), 550–62. https://doi.org/10.1016/j.
jaci.2019.10.031
Jeffs, R. (2011). MicroRNA and mRNA visualization in differentiating
C1C12 cells. Retrieved 22 March 2020, from https://commons.
wikimedia.org/wiki/File:MicroRNA_and_mRNA_visualization_in_
differentiating_C1C12_cells.jpg
Lentivirus.aspx
September 2020 Yale Scientific Magazine 9
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