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Coronavirus proteins and their directed evolution to inhibit virus replication

Abdulsattar, B. O. (2017) Coronavirus proteins and their directed evolution to inhibit virus replication. PhD thesis, University of Reading

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Coronaviruses are enveloped, positive sense, RNA viruses that infect many species of animals, including humans. Of the six coronaviruses that can infect humans, SARS-CoV and MERS-CoV are the etiological agents of most concern currently. Coronaviruses possess the most complex and largest RNA genomes among all RNA viruses. The genome contains up to 15 genes with multiple open reading frames (ORFs) encoding both structural and non-structural proteins. Coronaviruses encode about 30 proteins that play specific, and often essential, roles in viral replication and assembly. This thesis presents work done to express Murine hepatitis virus strain A59 (MHVA59) proteins such as Nucleoprotein and membrane genes, non-structural proteins (5,6,7,8,9,10,16) from gene1, part of non-structural proteins (Plpro, Y-domain from nsp3 and N-terminal from nsp12) and RdRp from C-terminal part of nsp12 in E. coli BL21 cells and mammalian 17clone-1 cells, the latter of which are permissive for MHV-A59. The efficiency of transfection and expression of the proteins in mammalian cells was evaluated. SUMOStar (small ubiquitin-like modifier) fusion technology was used to enhance protein expression in the eukaryotic system. Expressed proteins were detected by Western blot with an anti-His tag antibody. The ability of virus-expressed proteins to interfere with virus infection was tested and an inhibitory effect was detected by plaque assay. The coronavirus nucleoprotein (N) is an important component for both viral replication and transcription. Error-prone PCR (ep-PCR) was used with the N protein as template to introduce random error and the number of mutations introduced was calculated after 100 random colonies were sequenced to validate the mutagenesis. Transient expression of N protein was shown to increase the efficiency of infection and virus yield. The function of N was investigated by screening for dominant-negative N mutants, using a library of N variants constructed using ep-PCR. The cytotoxicity of N variants was tested by MTT assay. Expressed N variants showed a range of effects ranging from a 10-fold increase in virus yield associated with the wild type N to 10-fold inhibition of virus growth. One particular N variant, mut38, was non-toxic, but reproducibly inhibited virus growth. The potential to screen for dominant-negative N variants using cell survival was also assessed using different N libraries. The thesis also investigated different strategies aimed at purification of non-structural protein 16 (nsp16). The overall findings suggest an ability of virus-expressed proteins in eukaryotic cells to interfere with virus infection and demonstrate that such antiviral activity can be generated by mutating an important viral protein.

Item Type:Thesis (PhD)
Thesis Supervisor:Neuman, B. and Mulley, G.
Thesis/Report Department:School of Biological Sciences
Identification Number/DOI:
Divisions:Life Sciences > School of Biological Sciences
ID Code:73736


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