To assemble, or not to assemble: The initiation of retroviral capsid assembly

During the complex multistep process of retroviral maturation the immature Gag lattice is cleaved apart prior to the formation of the mature capsid shell. Proper maturation and capsid assembly are required for infectivity. The capsid protein (CA) contains two domains [N-terminal domain (NTD) and C-terminal domain (CTD)] and self-assembles to enclose the genomic RNA and other viral proteins. Retroviral capsids are highly polymorphic, exhibiting a wide-range of structures, and were previously modeled as a lattice consisting of 12 CA pentamers unequally distributed among 150--300 hexameric rings. Three CA-CA interfaces form between assembled subunits in the mature capsid: NTD-NTD, NTD-CTD, and CTD-CTD. Very little is known about the mechanism and pathway of assembly that form the mature capsid despite being attractive targets for antiviral therapy. A full understanding of events occurring during maturation, including capsid assembly will require further advances such as: (1) establishing how the cleavage products of Gag reorganize to stimulate capsid assembly during maturation, (2) defining how the CA subunits self-assemble, (3) characterizing the CA pentamer and its interaction with the previously characterized hexamer, and (4) visualizing the capsid lattice at an atomic resolution to guide inhibitor development. Understanding capsid assembly and structure will help to elucidate how the capsid interacts with other viral components to provide necessary functions during the early stages of viral replication.;Ancillary information suggests that the assembly of the capsid is nucleated at a specific location in the maturing virion following Gag cleavage. The presumptive nucleation event is explored in greater detail in this thesis using protein from Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) and a novel in vitro system that, at least for RSV, recapitulates many features of capsid assembly occurring in the virus. Results clearly demonstrate that RSV CA assembles via a nucleation-driven mechanism in vitro. Assembly was demonstrated to be electrostatically controlled and multivalent anionic salts were demonstrated to induce CA to form structures resembling authentic mature capsids. A complex as small as a CA dimer acted as a seed for assembly was demonstrated by isolating intermediates of the capsid assembly pathway. Biochemical evidence combined with genetic analyses supports the proposal that a similar nucleation step happens in the maturing virion.;The conserved major homology region (MHR) in the CTD was shown to be required for proper initiation of capsid assembly. Lethal MHR mutations were suppressed by secondary substitutions located in the NTD and CTD that increased the propensity of CA to assemble in vitro. The results are consistent with CA undergoing structural transformations during maturation. Proteolytic cleavage of Gag results in the accumulation of a transient CA-SP (spacer peptide, C-terminal of CA) intermediate. CA-SP enhanced the kinetics of in vitro assembly. The data suggest that CA-SP participates in nucleation of capsid assembly and the MHR participates in an early step of assembly.;Identifying icosahedral particles formed by multimerization of RSV CA protein permitted the development of a pseudo-atomic model for the retroviral CA pentamer. The data are the first description of all three mature CA-CA interactions (NTD-NTD, NTD-CTD, CTD-CTD) forming in pentamers and hexamers. Although further research is needed to understand the polymorphic nature of retroviral capsids, the results provide a major step forward in understanding the architecture of mature capsids at the atomic level. The data further support a model whereby initiation of capsid assembly involves a tripartite interaction between two NTDs and one CTD. The model invokes a critical participatory role not previously described for the interdomain NTD-CTD interaction in forming a functional retroviral capsid.