| Summary: | Viruses have played a central role in the evolution and ecology of cellular life since it first arose. The discovery of viral lineages that infect members of all three domain suggest that these lineages originated at the earliest stages of biological evolution. Research into these viruses is helping to elucidate the conditions under which life arose, and the dynamics that directed its early development. As well, investigations into viral molecular biology and ecological dynamics have propelled abundant progress in our understanding of living systems, including genetic inheritance, cellular signaling and trafficking, and organismal development. These aspects of viruses have motivated the current investigation into a novel, lysogenic variant of the archaeal virus Sulfolobus Turreted Icosahedral Virus (STIV), STIV3. Following previous work for STIV, a genetic system was developed for STIV3. This involved producing clones for five partitions of the viral genome. As well, an infectious clone was constructed using PCR and the Gibson assembly. Electroporating the infectious clone into pH shocked host cells produced free virions. Subsequent isolation of these virions in cell-free supernatant were applied to a fresh host culture, which, again, produced free virions. Cultures similarly infected with the original virus stock did produce a higher viral titer, but the timing of viral burst was highly coupled between the two. Bioinformatic investigations were also completed on STIV3, its sister variants STIV and STIV2, and metagenomic sequences collected from Yellowstone hot springs. 33 open reading frames were predicted for the 17,106bp STIV3 genome, all of which with homologs in at least one of the two other variants. Overall, the STIV3 genome resembled STIV more than STIV2 according to genetic distance, gene homology, and evolutionary signatures. dN/dS analysis of the turret-forming genes, A224, C384, and A586, revealed positive selection signatures that may be attributed to virus-host coevolution or host range expansion. The putative integrase gene B510 appears to be highly conserved among the STIV variants, particularly at the catalytic residues in the C-terminal. All other homologs exhibited high sequence similarity, indicating strong negative selection across most of the genome. Analyses were also carried out on metagenomic sequences, which found much higher sequence diversity in the environment than among STIV variants, even between STIV3 and STIV2, the latter of which was isolated from a hot spring in Iceland. Even with the small sample sets available, some population structuring was observed with PCA analysis. The work performed in this project provides a strong foundation for future investigations with this virus. A genetic system has been successfully constructed and partially validated. Future work can exploit this system to further our knowledge of virus biology, especially when combined with host molecular tools. As well, the STIV3 genetic system has the potential to further progress in biotechnology and bioengineering, particularly with the confirmed presence of a functioning integrase gene. National Science Foundation BioTiER scholars program
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