Discovering New Chlorovirus Glycoforms Using Polyclonal Antibodies

Tiffany Do Author

07/28/2021 Added

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Chloroviruses have a unique process of glycosylation and are the first viruses to encode most, if not all, of the machinery to glycosylate their proteins. This research was focused on the discovery of new N-linked glycan structures using polyclonal antibodies made against a chlorovirus with known glycan structures.

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[00:00:00.240]Hello. My name is Tiffany.
[00:00:01.410]And this summer I've been screening for new chlorovirus
[00:00:04.020]glycoforms using polyclonal antibodies.
[00:00:09.420]What are chloroviruses?
[00:00:11.400]Chloroviruses are these large plaque-forming viruses that have double-strand DNA
[00:00:15.770]and infect chorella-like green algae.
[00:00:18.270]These viruses have a genome of 290 to 370 kilobases.
[00:00:23.220]The PBCV -1 virus has 331 kilobases and encodes for approximately 410
[00:00:28.140]proteins.
[00:00:32.270]Chloroviruses have a unique process of glycosylation and are the first viruses
[00:00:36.890]to encode most, if not all of the machinery to glycosylate their proteins.
[00:00:42.230]Other viruses have structural proteins that are glycosylated and use host-encoded
[00:00:46.520]glycosylation machinery to modify proteins.
[00:00:51.900]Chloroviruses also have a sugar-coated outer protein shell on the virus
[00:00:56.160]where glycans are found.
[00:01:00.840]The PBCV-1,
[00:01:01.950]viron packages 148 unique virus-encoded proteins.
[00:01:07.230]The major capsid protein is what is glycosylated and makes up to about 40%
[00:01:12.120]of total viron proteins.
[00:01:16.110]Figure one is an image of the reconstruction of the PBCV-1 chlorovirus.
[00:01:24.040]What is glycosylation?
[00:01:25.750]Glycosylation is a post-transitional modification where sugar molecules are
[00:01:29.200]covalently attached to specific amino acids on a target protein.
[00:01:33.850]Glycosylation is complex because there are many enzymatic steps involved.
[00:01:37.900]Glycosylated proteins, also known as glycoproteins are found in all living
[00:01:41.470]organisms and play an important role in cell functions.
[00:01:50.920]Glycosylation increases diversity of the proteome because almost every
[00:01:54.610]aspect of glycosylation can be modified.
[00:01:57.400]This includes glycosidic linkage, glycan composition,
[00:02:01.270]glycan structure and glycan length. To discover new glycan
[00:02:05.800]structures, a study employing an antibody against type chlorovirus
[00:02:09.580]species PBCV-1 was designed.
[00:02:14.200]We assumed that viruses possessing a unique glycan structure would not react with
[00:02:18.790]an antibody made against PBCV-1 virus because it was established previously
[00:02:23.830]that anti-chlorovirus antibodies react mainly with glycans of the major capsid
[00:02:28.780]protein.
[00:02:30.340]This allows us to screen viruses for new glycan structure
[00:02:34.090]using antibodies made against chloroviruses with known glycan structures that
[00:02:38.200]are seen on figure two.
[00:02:42.220]If a virus does not get neutralized by the anti-PBCV-1 virus antibody,
[00:02:47.020]it is an indication of a possible new and unique glycan structure.
[00:02:51.850]Our hypothesis is that viruses will react differentially with antibodies made
[00:02:56.230]against PBCV-1. In figure two.
[00:03:00.650]The top panel depicts the unique structures of PBCV-1 major capsid protein
[00:03:05.150]glycans.
[00:03:06.410]A through D represents the different glycoforms of PBCV-1 E is the common
[00:03:11.330]common glycan core structure found in chloroviruses. F shows the legend of the sugar
[00:03:15.740]attachments on the glycans.
[00:03:21.420]The bottom panel represents the currently known glycan structures in chloro-
[00:03:24.690]viruses. Chlorovirus
[00:03:27.030]glycan structures are unique in that they have glucose attached to Asparagine
[00:03:31.200]rather than an amino sugar, such as acetylgalactosamine
[00:03:35.280]or acetylglucosamine.
[00:03:37.320]They also differ from each other in sugar composition and sequencing in the
[00:03:41.550]glycosylation sites.
[00:03:45.670]We tested our hypothesis with virus concentrations mixed with a serum of
[00:03:50.650]antibodies against PBCV-1.
[00:03:53.350]This was then incubated at room temperature and serial diluted from 10^-1
[00:03:57.280]to 10^-7. Samples are then plaque assayed to
[00:04:02.260]count infectious virons. We use untreated viruses for our control.
[00:04:11.680]The bottom circle represents the chlorovirus plaques on the lawn of green algal
[00:04:15.520]cells. As you can see, the control form plaques, while
[00:04:18.970]one of the antibodies formed no plaques,
[00:04:22.720]However, plaques can form when there is antibodies
[00:04:25.720]which could be due to unique glycan structures that won't react.
[00:04:31.700]Figure three represents the results of the neutralization of the
[00:04:36.080]Thirty-four chloroviruses tested by antibodies made against the PBCV-1 virus.
[00:04:41.450]The asterisk represents the viruses other than the PBCV-1 virus, that
[00:04:46.400]have known glycan structures.
[00:04:50.240]Thirty-four chloroviruses were screened for new glycoforms
[00:04:53.600]using an antibody made against PBCV-1 with known glycan structures.
[00:05:01.550]We found that viruses reacted differentially with antibodies made against PBCV-1.
[00:05:05.840]Therefore, our hypothesis was supported.
[00:05:12.590]Chloroviruses with resolved glycan structure, that have distinct glycoforms from
[00:05:17.030]PBCV-1
[00:05:17.570]glycans showed virus neutralization patterns varying from 0 to
[00:05:22.250]57%.
[00:05:26.580]Seven of the thirty-four assayed viruses are completely neutralized with the
[00:05:31.170]antibodies made against PBCV-1 virus.
[00:05:34.020]This means that the viruses have a glycan structure that resembles PBCV-1
[00:05:38.040]glycans.
[00:05:41.580]Applying a 50% of neutralization as the cut off value,
[00:05:44.970]We also identify twenty-four virus strains that are perspective candidates for following
[00:05:49.860]glycan composition and structure analysis
[00:05:52.560]using nuclear magnetic resonance (NMR) spectroscopy.
[00:05:57.900]To end,
[00:05:58.350]I'd like to give special thanks to the National Science Foundation for funding
[00:06:01.920]this research and to the University of Nebraska-Lincoln Summer Research
[00:06:05.700]Program for their support.

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Discovering New Chlorovirus Glycoforms Using Polyclonal Antibodies

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