Yang Guo:
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Viruses infecting hyperthermophilic archaea have gained wide attention during recent years owing to its remarkable diversity on morphology and genome structures. Although a substantial work was made to decipher the functions of the unique proteins encoded by archaeal viruses and to characterize the relationship of the viruses and host cells, the knowledge on the biology of the archaeal viruses is still limited. The crenarchaeal virus Sulfolobus islandics rod-shaped virus 2 (SIRV2), was emerging as a promising model for genetic and biochemical studies as well as for the characterization of different stages in viral infection cycle. However, similar to other archaeal viruses, the majority of the SIRV2 genome sequence showed little similarity to the public databases, which hindered the virus functional researches and raised challenges in protein comparison and prediction.
This thesis comprises two parts of results. Firstly, the functional characterization of a highly conserved operon of SIRV2 was described, revealing their unique protein structures, biochemical activities as well as possible biological process they may participate in. In the second part, the genome wide regulations of two Sulfolobus sofataricus P2 transcription regulators upon SIRV2 infection were firstly constructed.
A SIRV2 gene operon (gp17, gp18 and gp19) was found to be the only and highly conserved gene clusters in rudiviruses and filamentous viruses, suggesting an important function in both viral families. The experimental results showed that ORF131b (gp17) was a novel ssDNA binding protein, without a canonical ssDNA binding domain. A few positively charged residues forming a U-shaped binding channel on the gp17 dimer are crucial for its ssDNA binding activity. The intrinsically disordered C-terminus of gp17 was demonstrated to be involved in the interaction with gp18, which was predicted previously as a helicase but showed a ssDNA annealing activity in this study. gp19 was shown to possess a 5´ to 3´ ssDNA nuclease activity, in addition to the previously demonstrated endonuclease activity, and a weak interaction between gp18 and gp19 was also detected. The functional characterization of the entire operon and the strand-displacement replication mode proposed previously for SIRV2 strongly point to a role of the operon in genome maturation and/or DNA recombination in viral gene DNA replication and repair.
Two transcription regulators sso2474 and sso10340 from Sulfolobus solfataricus P2 were differently expressed upon SIRV2 infection. A method similar to, but simpler than, Chromatin immunoprecipitation combined with subsequent high-throughput sequencing (Chip-seq) was applied in this study to get insight into the gene composition of the two protein regulons in vivo. After mapping the sequence data with the genomes of Sulfolobus solfataricus P2 and SIRV2, protein sso2474 was detected to have a high binding affinity to virus genome by an unknown mechanism, whereas sso10340 or its interacted protein preferred to bind and regulate the host genes on several binding sites. A total of 27 enriched DNA fragments extracted from sso10340 complex were selected as candidate binding targets from the host genome for the further analysis using EMSA (Electrophoretic mobility shift assay) and foot printing assay. A palindromic sequence motif was defined based on the enriched sequences, and most of these target genes were involved in energy metabolism, transport and amino acid metabolism. The genome-wide binding profile presented here reflected two different kinds of regulon conditions and contribute to the knowledge expansion of the transcription regulation upon virus infection in Sulfolobus.