ANALYSIS OF THE ROLES OF THE P10 DOMAIN OF GAG AND OF DIMERIZATION IN ROUS SARCOMA VIRUS ASSEMBLY
Phillips, Judith Marie
In the Rous sarcoma virus (RSV) Gag protein, the 25 amino acid residues of the p10 domain immediately upstream of the CA domain are essential for spherical immature particle formation. The significance of this finding was not known at the inception of this work. I performed a systematic mutagenesis on this region and found excellent correlation between the amino acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. I then introduced exogenous cysteine residues that were predicted to form disulfide bonds across the dimer interface. Upon oxidation of immature particles a disulfide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer performed by a collaborator, Paul Murray, indicates that the N-terminal domains of CA must expand relative to the modeled mature hexamer to accommodate the p10 contact. Replacement of the NC domain of HIV-1 Gag with an artificial dimerization domain has been shown to support assembly of virus-like particles in vitro and in cells. Similar systems for RSV Gag have yielded inconsistent results, suggesting that the requirements for RSV assembly may differ from those for HIV-1 assembly. I attempted to clarify these requirements in native avian cells and in vitro using chimeric Gag proteins in which NC was replaced by a leucine zipper domain or by an inducible dimerization domain based on the FK506-binding protein (FKBP). Budding of the Gag-leucine zipper fusion protein from avian cells was observed but was not entirely consistent, and similar fusion proteins failed to assemble in vitro. The Gag-FKBP fusion proteins similarly failed to respond to the induction agent. I concluded that further characterization of Gag dimerization and of the sequence upstream of NC will be required before an assembly system based on artificial dimerization can be developed for RSV Gag.
retrovirus; assembly; Rous Sarcoma Virus
Previously Published As
EMBO J. 2008 May 7;27(9):1411-20
dissertation or thesis