Oh, Bonnie
(2015)
Multiple Functional Roles of the Bacteriophage HK97 Delta Domain in Capsid Assembly.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
The bacteriophage HK97 capsid assembly and maturation pathway has been well studied. Despite this, little is known about the initial steps of assembly. How do 415 capsid proteins, ~120 copies of protease protein, and 12 copies of portal protein interact to form a correctly sized and shaped capsid? Most dsDNA phages and herpesviruses require the use of separate scaffolding proteins in order to assemble properly. HK97 does not have a separate scaffolding protein; instead it has a 102 amino acid N-terminal extension of its major capsid protein, called the delta domain. Like other well-known viral scaffolding proteins, the delta domain is present during initial assembly but is absent in the mature structure, is mostly -helical in nature, has a high propensity of forming coiled coil interactions, and is flexible. We hypothesize that the delta domain is playing a scaffolding-like role in capsid assembly. Because the scaffolding-like region is actually part of the capsid protein, studying how a scaffolding protein contributes to capsid assembly is easily achievable using HK97. Using mutational analyses in a plasmid based system, I have tested the effects of delta domain point mutations, small insertions, and deletions on capsid assembly. Even single point mutations can cause assembly defects, rendering the capsid protein nonfunctional. Using biochemical and genetic assays, I have shown that different regions of the delta domain play different roles in capsid assembly. The HK97 delta domain is a multifunctional protein that is important for capsid protein solubility and expression, capsid shape, capsid size, protease incorporation, and portal incorporation. The flexibility and structure of the delta domain plays an important role in its ability to regulate the assembly process. Learning about how the delta domain interacts with different proteins during assembly provides a greater understanding of how a single protein functions in order to build macromolecular complexes. Important roles and regions identified in the HK97 delta domain are applicable to other related phage and herpesvirus scaffolding proteins, providing insight on the biological processes and requirements of viral capsid formation.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
27 September 2015 |
| Date Type: |
Publication |
| Defense Date: |
29 June 2015 |
| Approval Date: |
27 September 2015 |
| Submission Date: |
13 August 2015 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
197 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Integrative Molecular Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
bacteriophage, delta domain, capsid assembly, HK97 |
| Date Deposited: |
27 Sep 2015 23:45 |
| Last Modified: |
15 Nov 2016 14:29 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/25987 |
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