Retroviral capsid maturation: Contributions of the CA C-terminal domain

Maturation of virions is a critical and obligatory step toward infectivity for orthoretroviruses. Retroviruses initially bud from infected cells as immature particles. The structural proteins are synthesized as the Gag and Gag-Pol polyproteins. Concomitant with budding, the viral protease (PR) becomes active and cleaves Gag and Gag-Pol into the constituent proteins. Cleavage induces significant morphological changes to the interior of the virus, the most visually stunning aspect of maturation. Whereas the immature particle is roughly spherical, the mature virus contains a capsid of variable shape surrounding two copies of the viral positive-stranded RNA genome and associated proteins.;The mature capsid is made of the capsid protein (CA), which consists of an N-terminal domain (NTD) and C-terminal domain (CTD). The mature capsid is made of a hexameric lattice of CA punctuated by 12 pentamers variably located. The best studied CA-CA interaction is the dimerization interface between two CTD molecules. The dimerization interaction holds neighboring hexamers together and is generated by both hydrophobic and electrostatic interactions between the 310 helix and the second alpha helix of the CTD (alpha9). Further CTD-CTD stabilization is provided by the three-fold CTD interface which holds three hexamers together via the final alpha-helix (alpha11).;Much less is understood about the immature Gag particle. The size and flexibility of the domains of Gag have precluded successful x-ray crystallography and nuclear magnetic resonance imaging of Gag. The immature lattice is made exclusively of hexamers and forms spherical particles by insertion of gaps into the lattice. Recent cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET) of assembled truncated Gag molecules has provided models for immature assembly. The interhexameric dimerization interface is also present in immature particles but is predicted to look different than that of the mature capsid.;Using Rous sarcoma virus (RSV), we tested the new structural model of the immature interhexameric dimer interface by screening residues predicted to be at the interface. The failure of some of the alanine-substituted Gag proteins to assemble provides the strongest support to date that the current immature CTD dimer model is representative of the Gag lattice. Three of the mutants tested allowed Gag assembly but altered in vitro assembly of CA consistent with predictions from structural models. In total, these data support the hypothesis that the immature and mature dimers are different and regulated by distinct residues.;A novel mature CTD-CTD interaction was recently described for the three-fold axis of symmetry. Mutagenesis of RSV T214, selected for its potential influence at several stages of retrovirus production, provided support that the residue is critical for assembly of the mature capsid. Alteration of capsid stability for T214 mutants and the location of the residue in the loop between alpha10 and alpha11 of the CTD are consistent with structural models of the mature three-fold interface.;CA-SP, the capsid protein cleavage intermediate, assembled into the same types of particles as CA, albeit faster, and was able to nucleate CA assembly suggesting that CA-SP could be the first stage at which mature capsid assembly occurs. The intermediate may also serve as a nucleation point for capsid assembly. NMR analysis of the CTD-SP protein pointed to a potential close association between SP and the major homology region (MHR) and alpha9 of the CTD. The interaction is consistent with mutagenesis of CA in which SP mutations are able to restore infectivity to non-infectious MHR mutant viruses and the identification of maturation inhibitor escape mutants in HIV-1 that map to MHR residues.;Reported maturation effects by MHR and SP mutations pointed to role for these two regions in maturation regulation. The results presented in this dissertation support a model in which the capsid protein intermediate CA-SP establishes mature contacts in an SP-mediated fashion. In this model, residues of SP transiently interact or closely associate with the MHR and alpha9, causing slight conformational changes that break immature interactions and generate mature contacts in both the dimer and three-fold interface. By this mechanism, the entire CTD and SP coordinate with each other to regulate the outcome of maturation.