Instigate Interferon Alpha Signaling in Zika Virus Infected Placental Cells

Swetha Ashokkumar Author

07/27/2021 Added

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My research project is about instigating interferon alpha signaling (which is a innate immune system response to viral infections) in Zika virus infected cells. Additionally I tested to determine if treatment of Palmitoleate, an omega monosaturated fatty acid, helped to instigate the interferon alpha signaling in Zika virus infected cells.

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[00:00:00.600]Hello everyone. I'm Swetha Ashokkumar from the redox biology program. This summer,
[00:00:05.220]I was working in
[00:00:06.420]Dr.Natarajan's lab and my research project was to instigate interferon,
[00:00:10.530]alpha signaling in Zika virus,
[00:00:12.660]infected placental trophoblasts or placental cells.
[00:00:16.230]I would like to first give you some information about Zika virus.
[00:00:20.040]It is transmitted by the Aedes species mosquito. It is also a positive sense,
[00:00:24.750]single stranded RNA virus,
[00:00:26.670]meaning it can directly translate into viral proteins by the host cell.
[00:00:30.960]And currently there is no vaccine or approved medicine for Zika virus infection.
[00:00:36.390]One of the devastating effects of Zika virus is the congenital Zika syndrome.
[00:00:41.340]This happens when the mother is infected with Zika virus and transmits the virus
[00:00:45.570]through the placenta to the fetus.
[00:00:48.510]Microcephaly is a major birth defect of congenital Zika syndrome.
[00:00:52.830]It occurs when the baby's brain size is smaller than average,
[00:00:56.520]which causes damage to the brain and leads to many other defects in the infant,
[00:01:01.200]such as ocular damage, hearing impairments,
[00:01:04.050]difficulty swallowing breathing and moving it's limbs and body.
[00:01:08.880]Previous studies in.
[00:01:09.990]Our lab show that Palmitoleate and omega mono saturated fatty acid
[00:01:15.060]prevents Zika virus induced apoptotic,
[00:01:17.610]Cell death in placental trophoblast Palmitoleate treatment to Zika virus
[00:01:22.590]infected cells have also shown decreased viral RNA copies and
[00:01:27.300]increased cell viability. With the previous studies done in the lab,
[00:01:32.100]I can hypothesize that Palmitoleate prevents Zika virus infection in
[00:01:36.600]placental trophoblast by activating interferon alpha signaling.
[00:01:41.850]So what is interferon.
[00:01:43.320]Alpha signaling? Interferon alpha is a cytokine produced by the cells of the
[00:01:48.180]innate immune system in response to viral infection
[00:01:52.380]Here you can see an interferon alpha binds to a dimeric receptor
[00:01:57.300]Interferon alpha receptor 1, interferon alpha receptor 2, on the cell surface.
[00:02:02.580]This activates TYK2 and JAK1,
[00:02:05.820]which further activates transcription factors STAT1 and STAT2,
[00:02:10.470]that bind to IRF9 and translocate into the nucleus.
[00:02:15.840]This trimeric complex then binds to the interferon sensitive response
[00:02:20.610]element, which expresses interferon stimulated genes, such as ISG15,
[00:02:25.500]USP18, OAS1 and many others.
[00:02:29.460]USP18 has many functions, but here specifically it has two functions.
[00:02:35.310]USP18,
[00:02:36.780]Interacts with STAT2 and interferon alpha receptor 2 complex.
[00:02:41.880]When it binds to this complex,
[00:02:44.280]it stops interferon alpha signaling from occurring.
[00:02:48.570]The second function of USP18.
[00:02:50.070]Is that it cleaves the isopeptide bonds of ISG15,
[00:02:55.080]which is also an interferon stimulated gene,
[00:02:58.320]Once USP18 cleaves, the isopeptide bonds of ISG15,
[00:03:03.370]it inhibits it from binding to it's target proteins.
[00:03:08.320]So my question is can Palmitoleate activate interferon alpha signaling?
[00:03:13.780]So my approach to answer this question was to first infect JEG-3
[00:03:18.340]cells or placental trophoblasts or placental cells with 0.1
[00:03:22.960]MOI or multiplicity of infection, which means for every 10 cells,
[00:03:28.000]there was one virion that infected them.
[00:03:31.300]We infected my cells at a 24 hour and a 48 hour time point
[00:03:36.130]and treated them with interferon alpha the last four hours.
[00:03:39.460]During each of those time points,
[00:03:42.010]We then measured the expression of interferon stimulated genes using
[00:03:46.930]Q RTPCR or real-time PCR and used
[00:03:51.940]Western blot to measure the activation of STAT1 and USP18.
[00:03:57.940]Here, you can see.
[00:03:58.810]The mRNA expression of ISG15.
[00:04:02.080]If you notice that the interferon stimulated and Palmitoleate treated groups are
[00:04:06.370]expressing high levels of ISG15 in both the 24 and the 48.
[00:04:11.350]hour time points.
[00:04:13.030]Though there is- Though
[00:04:14.650]The ISG 15 is expressed.
[00:04:17.290]Relatively low in Palmitoleate
[00:04:19.810]treated group and interferon stimulated group alone.
[00:04:23.710]It is still expressed in a high quantity in interferon stimulated and Palmitoleate
[00:04:28.600]treated groups.
[00:04:32.310]OAS1 is an interferon stimulated gene as well.
[00:04:35.640]And it also has a similar pattern to ISG15.
[00:04:39.360]It is present higher.
[00:04:41.280]It is present in higher levels in interferon stimulated and Palmitoleate
[00:04:45.980]treated groups in 24 and the 48 hour time points.
[00:04:50.970]But in the 48 hour time points, you can see a major increase of OAS1
[00:04:55.470]in Palmitoleate treated group only
[00:05:02.400]expression of USP18 does not follow the pattern of previous
[00:05:07.380]data. You can see in the 24 hour time point,
[00:05:10.890]interferon stimulated group is expressing a high quantity of USP18.
[00:05:16.140]This is expected since stimulation of interferon alpha will produce high
[00:05:20.700]quantity of USP18,
[00:05:23.430]but you can notice that there's a decrease in USP18 when it's
[00:05:28.380]stimulated in interferon alpha,
[00:05:30.030]but also treated with Palmitoleate. In the 48 hour time
[00:05:34.800]point,
[00:05:35.070]you can notice an increase in expression of USP18 and
[00:05:39.990]Palmitoleate treated group and interferon stimulated group.
[00:05:43.590]But you can see a major increase in USP18 in the Zika virus infected group.
[00:05:48.930]This happens because of the role of the USP18,
[00:05:52.260]as it starts to inhibit interferon alpha signaling once interferon alpha is
[00:05:57.350]inhibited it causes the Zika virus infection to increase,
[00:06:01.190]which leads to increase quantity of USP18 present in Zika virus infected group.
[00:06:06.650]Now we will look at the activation of STAT1
[00:06:11.120]Activation.
[00:06:11.930]Of STAT1 indicates the activation of interferon alpha signaling.
[00:06:16.070]If you notice an interesting point here,
[00:06:18.350]you can see that the activation of phospho stat one is present in
[00:06:23.360]Palmitoleate treated group without any stimulation of interferon alpha,
[00:06:28.550]Though, there are thicker bands present interferon stimulated groups,
[00:06:32.120]It's quite interesting to see a slightly thicker or presence of phospho
[00:06:36.950]stat activation in Palmitoleate treated group without any interferon
[00:06:41.780]stimulation. If you see the activation of USP18,
[00:06:46.490]it is quite highly activated in Zika virus infected lane.
[00:06:50.720]This is because the one of the role of USP18 is to inhibit interferon
[00:06:55.670]alpha signaling.
[00:06:56.900]This would cause the Zika virus infection to increase leading a higher amount of
[00:07:01.430]activation of USP18 in Zika virus infected lane.
[00:07:06.110]But if you see the interferon stimulated and the Palmitoleate treated
[00:07:10.040]group here has lower amount or lower level of activation of USP18
[00:07:15.050]and even in the Palmitoleate treated group,
[00:07:18.860]you can notice a decrease in activation of USP18 when you compare it to the
[00:07:23.660]Zika virus infected lane
[00:07:26.120]This means that Palmitoleate treatment decreases the
[00:07:30.770]activation of USP18.
[00:07:34.550]So to conclude increased levels of interferon,
[00:07:37.760]stimulating genes are observed in both interferon alpha and Palmitoleate
[00:07:42.690]treated groups, activation of STAT1
[00:07:45.620]With only the treatment of Palmitoleate was observed,
[00:07:49.550]which confirms the activation of interferon alpha signaling without additional
[00:07:53.960]interferon stimulation.
[00:07:56.030]Treatment of Palmitoleate decreases the levels of USP18
[00:08:00.770]and presence of USP18 confirms activation of interferon alpha signaling
[00:08:06.200]Decreased levels of USP18 is required for an enhanced interferon alpha
[00:08:11.180]signaling to occur.
[00:08:14.480]I would like to thank the REU program and the University of Nebraska and the
[00:08:18.830]Redox Biology center for giving me an opportunity to be here this summer.
[00:08:23.300]I would also like to thank my lab members and especially my PI,
[00:08:27.050]Dr.Natarajan for having me in their lab,
[00:08:29.570]as I got to learn many techniques and I'm able to connect dots and learn
[00:08:33.740]differently than before. Thank you all.

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Instigate Interferon Alpha Signaling in Zika Virus Infected Placental Cells

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