Controlling the replication of HIV-1 with a quadruplet codon in mammalian cells

Yan Chen Author

04/03/2021 Added

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Student research day presentation.

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[00:00:00.900]Hi, my name is Yan. I'm a graduate student from the department of chemistry.
[00:00:06.140]My advisor is Dr.Jiantao Guo
[00:00:07.730]My project is controlling the replication of HIV-1
[00:00:12.530]with a quadruplet codon in mammalian cells.
[00:00:16.340]I will introduce my project in five parts, background,
[00:00:19.790]objective, methods, results. And the conclusion
[00:00:24.560]first, I will start with the background.
[00:00:27.660]In natural, we have 61 sense triplet codons. They can decode 20 nature amino acids.
[00:00:32.990]In additional, there're also three stop codons.
[00:00:36.380]People are thinking about if we can assign unnatural amino acid,
[00:00:38.970]to these three stop codons, we can expand the genetic code.
[00:00:44.060]Also with unnatural amino acids,
[00:00:46.460]we can introduce novel function to the proteins.
[00:00:50.600]Next, I want to show you how we incorporate unnatural amino acids to the proteins.
[00:00:54.890]First, I will use triplet stop code on as an example,
[00:00:58.700]to incorporate unnatural amino acids,
[00:01:00.560]we need two important components tRNA and the tRNA synthetase enzyme.
[00:01:05.060]tRNA synthetase
[00:01:07.010]is a enzyme can add unnatural amino acids to its corresponding tRNA.
[00:01:11.870]In the translation process,
[00:01:13.490]tRNA charged with unnatural amino acids can recognize the stop codons
[00:01:18.080]The unnatural amino acids can be added to the growing peptides.
[00:01:22.070]eventually this unnatural amino acids can be incorporated to the proteins.
[00:01:28.490]Using quadruple codon to decode unnatural amino acids.
[00:01:32.150]This process is very similar. Again,
[00:01:35.300]we need tRNA and tRNA synthetase in this case, the tRNA will
[00:01:40.250]have a four nucleotides anti-codon loop and in the translation the tRNA can
[00:01:45.200]recognize the quadruplet codon.
[00:01:47.540]And unnatural amino acid can be incorporated to the protein eventually.
[00:01:51.050]With quadruplet codon,
[00:01:53.750]we can further expand that genetic code because we'll have 256
[00:01:58.610]available quadruplet codons to decode unnatural amino acids.
[00:02:03.050]My project focused on HIV-1.
[00:02:05.450]This is a virus which can damage the immune system. Up to now,
[00:02:10.010]multiple HIV-1 vaccine are in development. However,
[00:02:13.880]based on the safety concern, none of them have been tested in the clinical trials.
[00:02:18.980]One of the concern is how can we control HIV-1 replication?
[00:02:24.140]Our strategy is we can use unnatural amino acid incorporation.
[00:02:28.850]The objective of my project is we want to use unnatural amino acid incorporation
[00:02:32.730]to control the HIV-1 replication,
[00:02:37.910]our program can be a potentially applied to the development of a safer
[00:02:42.860]HIV-1 vaccine. To achieve this goal,
[00:02:47.270]we introduce this quadruplet codon UAGA in the HIV-1 genome.
[00:02:52.460]With the tRNA synthetase enzyme and the tRNA, this quadruplet codon can
[00:02:57.290]decode an unnatural amino acid.
[00:02:59.590]And the first condition, when the unnatural amino acid is in present.
[00:03:03.250]The HIV-1 genome can be fully translated and synthesize fully
[00:03:08.060]functional virus. This virus can be potentially developed
[00:03:12.970]as a safe vaccine to trigger the immune response in human beings.
[00:03:17.920]In the other condition, when unnatural amino acid in absence,
[00:03:21.310]the translation will stop at the position of this quadruplet codon.
[00:03:25.750]So there'll be no virus generated. In this way, we can control the virus replication
[00:03:30.340]with the unnatural amino acids.
[00:03:33.610]Another advantage of this system is quadruplet codon
[00:03:36.580]is more difficult to mutate back to sense codons,
[00:03:40.060]even the virus have a relatively higher mutation rate.
[00:03:44.890]Before we testing our control system, We also want to test
[00:03:48.040]if this engineered tRNA variant have a good decoding efficiency in
[00:03:52.450]mammalian cell. This is a tRNA variant
[00:03:55.600]our group previously developed from the bacteria platform.
[00:04:00.700]To test the tRNA decoding efficiency. We constructed this two-plasmid system.
[00:04:05.800]We use Green Fluorescence Protein as a reporter gene.
[00:04:09.340]We introduced a quadruplet codon in the middle of the GFP.
[00:04:14.110]If the tRNA have a good efficiency to incorporate unnatural amino acid in this position,
[00:04:18.760]we will observe a strong fluorescence intensity.
[00:04:23.620]And then we co-transfect these two plasmids to the mammalian cell Hek 293T.
[00:04:27.490]We first took microscope images.
[00:04:32.350]From here, We can clearly observe green fluorescence signals.
[00:04:35.740]We also run the flow cytometry analysis. Combine these two results,
[00:04:40.420]we confirmed that the tRNA variant-1 has the best decoding efficiency.
[00:04:45.220]because we can detect strong fluorescence intensity with this tRNA variant
[00:04:53.080]To test if the control system works well,
[00:04:55.330]we construct this plasmid. In this plasmid, it contains HIV-1 genome.
[00:05:01.150]And also we have introduced a quadruplet codon in the HIV-1 genome.
[00:05:06.610]And then, we transfer this plasmid to HEK 293T cells,
[00:05:10.960]When unnatural amino acid is in present.
[00:05:13.810]The HEK 293T cells can produce HIV virus.
[00:05:18.190]And then, we'll use the virus to infect a reporter cell.
[00:05:21.970]This reporter cell can produce luminance when they are infected by HIV virus.
[00:05:26.830]And in the other case, when unnatural amino acid is in absent.
[00:05:30.770]The HEK 293T cells
[00:05:32.730]can not generate any functional HIV-1 virus.
[00:05:37.240]In theory,
[00:05:38.080]we cannot detect any luminance from the reporter cells, because the reporter cells
[00:05:42.910]are not infected by the HIV virus.
[00:05:46.600]We tested two different position to introduce the quadruplet codons.
[00:05:51.130]This is the results of the infectivity assay.
[00:05:55.480]From the infectivity assay result,
[00:05:57.560]we confirmed that HIV replication is tightly controlled by the unnatural amino acids.
[00:06:02.240]because we can only observed strong luminance signals
[00:06:07.190]when the unnatural amino acid is in present in both positions.
[00:06:11.870]In conclusion, firstly,
[00:06:14.180]we evaluate an engineered tRNA in mammalian cells.
[00:06:19.370]And we have found a variant which have a very good
[00:06:22.460]decoding efficiency in mammalian cells.
[00:06:26.180]Secondly, we confirmed that we can control the virus
[00:06:28.700]replication tightly in mammalian cells with the quadruple codons.
[00:06:31.940]Our results can potentially apply to a safer
[00:06:36.470]HIV-1 vaccine development and help us to fight with this disease.
[00:06:42.590]Lastly, I want to thank my advisor,
[00:06:46.040]Dr.Jiantao Guo and my committee members.
[00:06:49.520]Also, I want to thank my current and former group members.
[00:06:53.960]Lastly, I want to thank my collaborators.
[00:06:56.360]They helped me to accomplish these project together. Thank you.

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Controlling the replication of HIV-1 with a quadruplet codon in mammalian cells

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