ROLE OF PROTEIN CONFORMATION AND CHARGE-SHIELDING IN RETROVIRAL CAPSID MATURATION
Open Access
- Author:
- Lokhandwala, Parvez Mubasshir
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 08, 2010
- Committee Members:
- Rebecca C Craven, Dissertation Advisor/Co-Advisor
Rebecca C Craven, Committee Chair/Co-Chair
John Warren Wills, Committee Member
Leslie Joan Parent, Committee Member
Ralph Lauren Keil, Committee Member
Jianming Hu, Committee Member - Keywords:
- MHR
maturation
capsid
CA
Retrovirus
Rous sarcoma virus - Abstract:
- Currently there is no cure available for an HIV-infected individual. The current anti-retroviral regimen that requires treating a patient indefinitely with multiple medications suffers from several disadvantages including emergence of revertant viruses that negate the effectiveness of drugs and adverse effects of the medications. Hence, there is a critical need to develop novel drugs to add to the anti-retroviral arsenal. The capsid maturation step in the retroviral life cycle is being explored as a potentially novel therapeutic target. However, to design safe and effective drugs, it is important to understand the molecular details of this process. Retroviruses bud as non-infectious immature virus that must undergo an essential process of maturation to become infectious. During this process, the immature shell formed by the Gag structural polyprotein is cleaved by the viral protease and the mature shell is reassembled by the newly released capsid (CA) protein. The retroviral capsids are predicted to assemble based on fullerene principles, primarily composed of hexameric arrays of the CA protein with twelve CA pentamers interspersed. The α1, α2 and α3 helices of N-terminal domain (NTD) of the CA protein associate to form a hexameric or a pentameric ring of CA protein that is connected to the adjacent hexameric ring via the dimerization of the second helix (dimer helix) in the C-terminal domain (CTD). Additionally, heterotypic NTD-CTD interactions also exist. An evolutionarily conserved region in the beginning of the CTD, known as the major homology region (MHR), plays multiple roles during virus life cycle including immature and mature virus assembly. However, based on the current models of mature retroviral capsids, the MHR motif does not appear to contribute directly and significantly to any of the CA-CA interfaces. To understand the function of the MHR during capsid maturation and to identify other regions of the protein involved in this process, we used a gain-of-function approach. We have isolated two substitutions in the dimerization helix of Rous sarcoma virus CA protein that have the ability to suppress lethal defects in capsid maturation imposed by mutations in the MHR motif just upstream. Together with two previously published suppressors, these define an extended region of the dimerization helix that is unlikely to contribute directly to CA–CA contacts but whose assembly-competence may be strongly affected by conformation. The broad-spectrum suppression and temperature-sensitivity exhibited by some mutants argues that they act through modulation of protein conformation. Cryo-electron microscopy analysis of a temperature-sensitive mutant produced at permissive and non-permissive temperatures provides morphological evidence of the role of MHR in capsid assembly. These findings provide important biological evidence in support of a significant conformational change involving the dimerization helix and the MHR during maturation. While the structural details of the presumptive CA-CA interactions occurring during mature capsid assembly are now becoming clearer, the mechanism by which this capsid assembly process is regulated remains unknown. Based on the ability to trigger in vitro assembly of HIV-1 and RSV CA using sodium chloride and sodium phosphate respectively, and the identification of a putative cluster of positively charged residues at the tripartite interface of RSV capsid, we hypothesized that electrostatic shielding of these charged residues is necessary to trigger capsid assembly. We describe spontaneous isolation of two charge-change mutations at the tripartite interface that rescue the infectivity of a previously characterized crippled MHR mutant that is assembly-incompetent. Several targeted mutations that reduce the positive charge at the tripartite interface rescue infectivity of the assembly-incompetent MHR mutant. Certain CA proteins with mutations that reduce the positive charge at the tripartite capsid interface exhibit enhanced assembly kinetics and reduced phosphate dependency as measured by the in vitro assembly assay. Together, our data provide genetic as well as biochemical evidence for a biological role of charge-shielding at the tripartite interface during capsid assembly, presumably during nucleation. The viral and cellular factors that may play such a role in situ during capsid assembly are discussed. We propose a model of capsid maturation wherein the conformational changes of CA protein and the screening of electrostatic forces at CA-CA interfaces play critical roles in control of capsid assembly.