Contributions of the Capsid N-terminal Domain to the Regulation of Assembly and Maturation in Rous Sarcoma Virus
Open Access
- Author:
- Heyrana, Katrina Joy
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 09, 2014
- Committee Members:
- Rebecca C Craven, Dissertation Advisor/Co-Advisor
Rebecca C Craven, Committee Chair/Co-Chair
Jianming Hu, Committee Member
Faoud T Ishmael, Committee Member
Clare E Sample, Committee Member
John Warren Wills, Committee Member - Keywords:
- retroviruses
capsid assembly
Gag assembly
virus maturation - Abstract:
- Retroviruses are pathogens with the unique ability to integrate directly into the genome of their host cells, creating permanent infections that are propagated with every subsequent cell division. The lasting nature of integrated proviruses makes retroviral infections such as HIV difficult to eradicate despite the availability of effective drug combinations that suppress viral replication. Thus, continued exploration of the retroviral life cycle remains essential to the identification of targets for future drug design. One attractive prospect for pharmacological intervention is the assembly and maturation of new viral particles. Since proper assembly and maturation are prerequisites for infectivity, agents that interfere at these stages could potentially prevent viral transmission. Immature retrovirus assembly is a complicated, multifactorial process that centers on the formation of an spheroidal Gag shell around two copies of the RNA genome. While Gag-Gag interactions in the shell occur between all the components of the polyprotein, the CA (capsid) protein mediates major contacts that determine proper spacing and successful assembly of a new virus. After immature particle release and protease activation, retroviruses undergo a maturation step that results in a complete reorganization of the viral interior, culminating in the assembly of newly liberated CA into a polyhedral capsid around the nucleocapsid (NC)-coated genome and reverse transcriptase (RT). CA sequence manipulation can interfere with both initial viral particle release and formation of the mature capsid. Therefore, careful characterization of the CA interactions that build immature and mature virions and identification of the regions of CA that regulate maturation is vital to our understanding of how to build an infection-competent virus. The flexible loop (FL) region of the CA protein poses one potential control point for the ordered assembly of infectious particles. Located on the N-terminal domain (NTD) of the two-domain CA protein, the FL is a structurally heterogeneous, sequentially divergent, and relatively dynamic stretch of residues. Mutations in the FL sequence alternately disrupts budding or maturation, and a number of host restriction factors bind the FL, suggesting a possible role for the region in particle assembly, the maturation transition, and even establishment of a new infection. The structural significance of the FL is further underscored by a recent study showing that the region participates directly in an interface connecting adjacent monomers of CA within an immature Gag lattice. In order to determine how the FL could participate in the assembly of an infection-competent virus, we undertook targeted mutagenesis to identify essential residues for viral infectivity. The strongest effects were seen at three charged residues, which were further characterized to differentiate whether mutations affected immature assembly, the maturation transition, or a combination of both steps. We found that charges at these positions strongly affected the regularity of initial particle formation, but their absence did not preclude in vitro formation of mature CA-CA contacts. Modeling of the residues into the immature Gag lattice structure suggested that these residues participated in the FL-FL interface, which is unique to the Gag lattice. A gain of function suppressor mutant that compensated for an infectivity defect at one of these positions mapped in the model to an extended form of the same interface, reinforcing the possibility that these charged residues primarily serve as control points in the formation of the immature Gag lattice. Based on these findings, we extended the current model of the immature Gag lattice to include the FL region, which was partially omitted in the published structures. Molecular dynamics modeling performed by collaborators at UIUC identified novel contacts in the FL-FL interface that we tested in the viral context. This work identified at least one completely new interaction that we assessed with dual cysteine mutations in an attempt to link monomers across the interface. Infectivity and particle release data with the double mutant showed aberrant Gag release and processing, providing some of the first viral support for the immature lattice model that appear to demonstrate interactions across the newly described FL-FL interface. Since published structures of the mature capsid do not suggest a direct structural role for the FL in any interfaces in the lattice, we also examined how FL residues may work cooperatively with distant regions of the CA protein. Using a triple mutant suppressor virus that required an FL and CTD mutation to compensate for an infectivity defect from a founder lesion in the N-terminus of CA, we characterized how these residues worked cooperatively to promote the assembly of a maturation-competent, infectious virus. Altogether, these data support a strong regulatory role for the FL in the assembly of a nascent viral particle, and suggest that proper control of Gag lattice formation facilitates subsequent reorganization into a mature capsid.