Assignment 2 - Coronavirus

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Jan 9, 2024

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Assignment #2 – Coronavirus 1. In your own words, please describe the main concepts being investigated. What is the context for this research? (i.e., is it an entirely new idea? Is it following up on previous research?) Explain. The main topic of this research is following a long thread of SARS-CoV-2 therapeutic research. It has been a challenge to create replicons for SARS-CoV-2 due to its large genome. In this paper the researchers are exploring the use of yeast-based reverse genetics system to create self-replicating spike-deleted SARS-CoV-2 RNA [1]. The purpose of creating these self-replicating RNA is to insert them into a large range of cell types in which they can generate replicon delivery particles. These replicons provide replication without producing the infectious virus which allows for smaller lower-containment labs to be able to research SARS-CoV-2. 2. In the paper, what method did the authors use to specifically investigate and characterize the main concept? Describe two other methods or techniques used for some of the other experiments described. Limit your description in this section to only 3 techniques total. Use your own words to describe the method, rather than a technical list of details. SARS-CoV-2 Replicon design and launch optimization: To assemble the replicon a newly published synthetic genomics platform was utilized. This system uses saccharomyces cerevisiae ability to recombine overlapping DNA fragments, this process is known as transformation-associated recombination (TAR) cloning [2].

TAR cloning [3] involves viral RNA is prepared from infected cells and used to amplify the overlapping DNA fragments using RT-PCR, these are then transformed into S. cerevisiae and clones are later screened for correct assembly. Trans-complementation of replicons with spike: BHK-21 cells were transfected with spike-expressing plasmid and after 24 hours these cells were coelectroporated [4] with spike-deleted mNeonGreen replicon RNA and N mRNA. Electroporation is a process in which high-voltage electricity is pulsed using a BTX electrosquare porator ECM 830 to introduce DNA into cells, this allows for immunofluorescence visualization using microscopy. Neutralization assays with RDPs: Huh-7.5 AT Cells are seeded into well plates and human monoclonal antibodies were serially diluted in plating media [1] then mixed with RDPs. This antibody-RDP mixture was then mixed, incubated, and applied to the Huh-7.5 AT cells. Cells were incubated and the supernatant was collected 24 hours later for the Gluc signal to be read and normalized. Cells were also fixed and stained for immunofluorescence of N protein. 3. Summarize the main findings / conclusions of the paper. The main findings of this paper were the several features of SARS-CoV-2 RDPs. First, RDPs can be used to, more efficiently, generate single-cycle SARS-CoV-2 virions for use in spike-directed research. These areas include screening for antibodies, spike variations, and spike mutations. Second single-cycle infectivity can be achieved using VSV glycoproteins as a packaging mechanism which provides a

mode of usage in which replicon delivery is ACE2-independent. Lastly this study found that RDPs are much more resilient compared to RNA transfection in terms of storage and distribution as they do not require any specialized equipment to use. This gives an opportunity for so many labs to study this as specialized equipment is incredibly expensive and inaccessible to many labs. In conclusion, this paper was able to prove that spike-deleted SARS-CoV-2 replicons give a much more convenient and accessible way to study SARS-CoV-2. 4. If you were continuing this research project, what experiments would you do next? Explain the rationale for WHY you would choose those experiments and briefly describe HOW you would do them . The spike protein of SARS-Cov-2 is the reason for the many mutations [5] we have seen regarding Covid 19. The ability to make these mutants noninfectious and much less specialized to work with allows for more testing to be performed. If I was continuing this research, I would use these SARS-CoV-2 spike-deleted replicons and RDPs for drug testing and vaccine development. Replicons have been used previously in drug production to assess efficacy and bioavailability [6]. I would also use these replicons as a possible vaccine platform. Replicon vaccines are already in the works [6], an example I would follow would be from a previous study in which a vaccine for cytomegalovirus was produced by purification of replicon particles [7] from cells co-transfected with a CMV replicon transcript to encode structural protein genes.

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Bibliography 1. Inna Ricardo-Lax1 †, Joseph M. Luna1 †, Tran Thi Nhu Thao2,3,4, Jérémie Le Pen1 , Yingpu Yu1 , H.-Heinrich Hoffmann1, et al. Replication and single-cycle delivery of SARS-CoV-2 replicons. Science.2021;374: 1099-1106 2. Thao T, Fabien Labroussaa, Ebert N, V’kovski P, Stalder H, Portmann J, et al. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature. 2020;582: 561-565 3. Kouprina N, Larionov V. Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology. PMCID. 2016;125: 621-632. PMID: 27116033 4. Potter H, Heller R. Transfection by Electroporation. PMCID. 2010;62:1-6. PMID: 18265334 5. Li Q, Wu J, Nie J, Zhang L, Hao H, Liu S, et al. The Impact of Mutations in SARS-CoV- 2 Spike on Viral Infectivity and Antigenicity. Cell. 2020;182(5):1284-1294. PMID: 32730807 6. Hannemann H. Viral Replicons as Valuable Tools for Drug Discovery. PMC. 2020;25(6):1026-1033. PMID: 32272194 7. Bernstein D, Reap E, Katen K, Watson A, Smith K, Norberg P, et al. Randomized, double-blind, Phase 1 trial of an alphavirus replicon vaccine for cytomegalovirus in CMV seronegative adult volunteers. Vaccine. 2009;28:484-493

Assignment 2 - Coronavirus

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