Presentation Title

Analyzing the effects of site-directed mutagenesis within the B345 C-terminal tail on ESCRT-III protein recruitment

Faculty Mentor

Jamie C. Snyder

Start Date

17-11-2018 2:30 PM

End Date

17-11-2018 2:45 PM

Location

C161

Session

Oral 3

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA archaeal virus that was isolated from an acidic (pH2) hot (82°C) spring within Yellowstone National Park (YNP). STIV infects Sulfolobus solfataricus; however, the mechanisms behind infection are still unclear. The overall goal of the lab is to develop the first complete replication cycle for STIV, with my project specifically focusing on delineating the cellular components required for assembly of STIV. During an STIV infection, an ESCRT (Endosomal Sorting Complex Required for Transport) gene cluster is up-regulated; and interestingly enough utilization of host ESCRT proteins for budding is a conserved pathway in eukaryotic viruses. An ESCRT-III protein along with the STIV major capsid protein (B345) and other viral proteins were found in the membrane subcellular fraction of S. solfataricus during infection leading us to further investigate the functional relevance of this apparent interplay between STIV and ESCRT machinery. A yeast two-hybrid assay further demonstrated the interaction between ESCRT-III (SSO0619) and B345. Our hypothesis is that B345 interacts with ESCRT-III, such that it recruits the ESCRT-III protein to vesicles that will eventually become the internal membrane of the assembled virion. My project entailed utilizing site-directed mutagenesis to change specific amino acids within the C-terminal end of B345 because this region plays a role in the attachment of ESCRT-III. Successfully created constructs will then be tested in context of virus replication, B345 alone in S. solfataricus cultures, and in co-immunoprecipitation assays. If STIV assembly utilizes ESCRT proteins, we should alter assembly when this interaction is disrupted.

Summary of research results to be presented

To analyze the functional interplay between host and viral proteins, a yeast two-hybrid analysis was conducted with S. solfataricus ESCRT components and STIV proteins associated to the formation of major capsid proteins (MCP). An interaction was observed between SSO0619 and B345, and this interaction was verified through a GST pull-down assay (Jamie C. Snyder et al., 2013).

The C-terminal end of B345 was mutated and the mutated construct was successfully transfected into Sulfolobus solfataricus. Protein expression was then induced through the use of an arabinose promoter. Future studies include running Western blot analyses to confirm protein expression and conducting co-immunoprecipitation assays.

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Nov 17th, 2:30 PM Nov 17th, 2:45 PM

Analyzing the effects of site-directed mutagenesis within the B345 C-terminal tail on ESCRT-III protein recruitment

C161

Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA archaeal virus that was isolated from an acidic (pH2) hot (82°C) spring within Yellowstone National Park (YNP). STIV infects Sulfolobus solfataricus; however, the mechanisms behind infection are still unclear. The overall goal of the lab is to develop the first complete replication cycle for STIV, with my project specifically focusing on delineating the cellular components required for assembly of STIV. During an STIV infection, an ESCRT (Endosomal Sorting Complex Required for Transport) gene cluster is up-regulated; and interestingly enough utilization of host ESCRT proteins for budding is a conserved pathway in eukaryotic viruses. An ESCRT-III protein along with the STIV major capsid protein (B345) and other viral proteins were found in the membrane subcellular fraction of S. solfataricus during infection leading us to further investigate the functional relevance of this apparent interplay between STIV and ESCRT machinery. A yeast two-hybrid assay further demonstrated the interaction between ESCRT-III (SSO0619) and B345. Our hypothesis is that B345 interacts with ESCRT-III, such that it recruits the ESCRT-III protein to vesicles that will eventually become the internal membrane of the assembled virion. My project entailed utilizing site-directed mutagenesis to change specific amino acids within the C-terminal end of B345 because this region plays a role in the attachment of ESCRT-III. Successfully created constructs will then be tested in context of virus replication, B345 alone in S. solfataricus cultures, and in co-immunoprecipitation assays. If STIV assembly utilizes ESCRT proteins, we should alter assembly when this interaction is disrupted.