The mechanisms of foamy virus capsid assembly.

摘要

The foamy viruses (FVs) possess a truly unique mode of replication among retroviruses. Several of the key differences in the assembly and morphogenesis of FVs allow for their potential use as vectors for gene therapy. The definition of the precise mechanisms used by FVs in the formation of infectious virus is therefore of considerable importance. In this study, I have examined the role of FV Gag in capsid assembly and viral particle budding.; First, I have shown that FV assembly occurs in the cytoplasm in a manner similar to the B and D-type retroviruses, through a domain akin to the cytoplasmic targeting/retention signal (CTRS). Unlike these retroviruses, however, FV Gag is not myristylated and therefore a disruption of the CTRS signal was found to abrogate particle assembly, due to the lack of an alternate targeting signal on Gag. I found that the addition of a plasma membrane targeting signal was able to restore assembly to a CTRS mutant, although these mutant viruses were not replication competent, indicating that the production of infectious virus is site-specific for the FVs.; In addition, I have performed a deletion mutagenesis of Gag and have discovered that a substantial portion of the central region of Gag is dispensable for the assembly of FV capsids and the production of infectious virus. This finding is in striking contrast to most other retroviruses that assemble capsids via Gag-Gag interaction domains located in the central capsid (CA) domain of the protein. Also, I have shown that a number of deletion mutations at the N-proximal region of Gag severely limit particle release, suggesting the presence of a Gag-Gag interaction domain in this region.; Finally, I have developed a novel assay to examine the protein-protein interactions occurring between Gag and Env that are required to mediate capsid budding. This assay involves binding studies utilizing a bacterially expressed and purified Env leader peptide fused to glutathione S-transferase (GST). Using this assay, I have found a specific interaction of capsids, but not free Gag protein, with the N-terminus of Env.