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Investigating the role of NS5A domain III in protecting the hepatitis C virus replication complex

Investigating the role of NS5A domain III in protecting the hepatitis C virus replication complex
Investigating the role of NS5A domain III in protecting the hepatitis C virus replication complex
As a positive sense RNA virus, the Hepatitis C virus makes use of a membrane enclosed replication complex (RC) to replicate its genome in a protected manner. As part of a separate project attempting to visualise the RC, a fluorescent peptide, clover, and a Dihydrofolate reductase (DHFR) destabilizing domain was inserted into the NS5A coding region of a JFH1 replicon. Unexpectedly it was found that the insertion site significantly influenced RNA replication. Under conditions where the DHFR domain was destabilized, insertion within domain III inhibited replication, while insertion at the carboxyl terminus of the protein was better tolerated. However, any difference in replication resulting from the choice of insertion site was lost when transfected cells were treated with trimethoprim to stabilise the DHFR domain. This suggested that rather than causing mis-folding of domain III, insertion into it disrupted an NS5A function that protects the protein from proteolytic degradation. The aim of this work was therefore to further characterise this activity.

In an effort to map the key regions of domain III involved, deletions were introduced into domain III of the replicon construct, within the Clover/DHFR fusion protein linked to the carboxyl terminus. Replication assays identified a 81 amino acid region within domain III of NS5A that seemingly protects these constructs during trimethoprim withdrawal. Further mapping of this region using a series of smaller overlapping deletions further narrowed this to a 61 amino acid sequence.

To confirm that this region of interest is indeed protecting NS5A from degradation, domain III was duplicated to be either full length or truncated. Replication assays with this constructs showed that an intact domain III is capable of protecting replicons from DHFR-mediated degradation. By scrambling domain III it was found that this activity if not dependent on the primary sequence, suggesting that it is due to an intrinsic property of domain III.

The final part of this work was to investigate potential benefits of domain III to the virus lifecycle, specifically investigating the impact of domain III truncation on IFN sensitivity and antigen presentation. However, several experimental issues hindered this work and it was not possible to confirm or deny a role for domain III in modulating these pathways.
University of Southampton
Sullivan, Edward David Kenneth
377a72d4-a765-4501-8656-0f1e22cd345e
Sullivan, Edward David Kenneth
377a72d4-a765-4501-8656-0f1e22cd345e
Mccormick, Christopher
0fce14bf-2f67-4d08-991f-114dd1e7f0bd
James, Edward
7dc1afb7-d326-4050-89fc-1f4e2a1a19a4

Sullivan, Edward David Kenneth (2017) Investigating the role of NS5A domain III in protecting the hepatitis C virus replication complex. University of Southampton, Doctoral Thesis, 258pp.

Record type: Thesis (Doctoral)

Abstract

As a positive sense RNA virus, the Hepatitis C virus makes use of a membrane enclosed replication complex (RC) to replicate its genome in a protected manner. As part of a separate project attempting to visualise the RC, a fluorescent peptide, clover, and a Dihydrofolate reductase (DHFR) destabilizing domain was inserted into the NS5A coding region of a JFH1 replicon. Unexpectedly it was found that the insertion site significantly influenced RNA replication. Under conditions where the DHFR domain was destabilized, insertion within domain III inhibited replication, while insertion at the carboxyl terminus of the protein was better tolerated. However, any difference in replication resulting from the choice of insertion site was lost when transfected cells were treated with trimethoprim to stabilise the DHFR domain. This suggested that rather than causing mis-folding of domain III, insertion into it disrupted an NS5A function that protects the protein from proteolytic degradation. The aim of this work was therefore to further characterise this activity.

In an effort to map the key regions of domain III involved, deletions were introduced into domain III of the replicon construct, within the Clover/DHFR fusion protein linked to the carboxyl terminus. Replication assays identified a 81 amino acid region within domain III of NS5A that seemingly protects these constructs during trimethoprim withdrawal. Further mapping of this region using a series of smaller overlapping deletions further narrowed this to a 61 amino acid sequence.

To confirm that this region of interest is indeed protecting NS5A from degradation, domain III was duplicated to be either full length or truncated. Replication assays with this constructs showed that an intact domain III is capable of protecting replicons from DHFR-mediated degradation. By scrambling domain III it was found that this activity if not dependent on the primary sequence, suggesting that it is due to an intrinsic property of domain III.

The final part of this work was to investigate potential benefits of domain III to the virus lifecycle, specifically investigating the impact of domain III truncation on IFN sensitivity and antigen presentation. However, several experimental issues hindered this work and it was not possible to confirm or deny a role for domain III in modulating these pathways.

Text
Edward D K Sullivan Final Thesis - Version of Record
Available under License University of Southampton Thesis Licence.
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Published date: September 2017

Identifiers

Local EPrints ID: 422266
URI: https://eprints.soton.ac.uk/id/eprint/422266
PURE UUID: 0513567c-a6fa-449b-9660-5b5d7b758930
ORCID for Christopher Mccormick: ORCID iD orcid.org/0000-0002-6155-9161

Catalogue record

Date deposited: 20 Jul 2018 16:30
Last modified: 13 Jun 2019 00:35

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Contributors

Author: Edward David Kenneth Sullivan
Thesis advisor: Christopher Mccormick ORCID iD
Thesis advisor: Edward James

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