Date of Award
Bachelor of Science (B.S.) in Biology and University Honors
SSV1, HiFi Assembly, Fusellovirus, Archaeal Virus, Mutagenesis
SSV1, spindle-shaped virus 1, is an archaeal virus with a unique shape that belongs to the family of fuselloviruses and infects hyperthermophilic archaea that thrive at 80°C and pH 3. SSV1 has two capsid proteins, VP1 and VP3, that presumably produce the lemon-shaped virion structure characteristic of fuselloviruses. The VP1 amino acid sequence is highly conserved among SSVs. Here, we investigate the importance of the proteolytic cleavage site in position E66 in VP1 by changing its glutamic acid to the other 19 amino acids. We have developed a new protocol for creating SSV1 mutants through HiFi Assembly (NEB) to increase the efficiency of mutagenesis and compile a library of SSV1 mutants. By optimizing this process --ensuring the original template is removed and that the total moles of DNA within the reaction lies below 0.2 picomoles--, we generated SSV1 mutants with the cleavage site of VP1 mutated to 16 other amino acids, demonstrating that HiFi Assembly can be used to successfully create a large number of mutations in a single reaction. We show that SSV1 mutant E66Q, with glutamine at the cleavage site, is viable and produces heterogeneous virion structure, while E66M, with methionine, produces virions similar to the wild-type. The characterization of the remaining mutants is ongoing.
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Liyanaarachchi, Thejanee, "Virology: Mutagenizing the Proteolytic Cleavage Site in the Major Capsid Protein in SSV1" (2021). University Honors Theses. Paper 1156.