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William M. Schneider
Rutgers, the State University
Graduate Program: Biochemistry and Molecular Biology
Thesis Advisor: Monica J. Roth
RWJMS - Room V-10
Friday, March 26, 2010
A pervasive theme in the post-genomic era is the quest to obtain three dimensional protein structures, providing insights into biological function. This thesis studies two stages in the retroviral lifecycle, virus-cell membrane fusion and integration, with a particular focus on the development and application of new protein production technologies.
The G541R mutation within the ectodomain of the MuLV TM protein was previously shown to be responsible for a unique phenotype relating to membrane fusion; reduced cell-cell fusion while virus-cell fusion is maintained. Through biochemical analysis it was discovered that an altered structural conformation of TM was responsible for this unique phenotype.
The MuLV integrase (IN) protein was studied to probe the mechanism of retroviral integration. MuLV IN encodes insertions in both the N-terminal (NTD) and C-terminal (CTD) domains that are not present in HIV and other retroviral INs. The MuLV IN NTD encodes an additional 50 amino acids prior to the conserved HHCC zinc-binding domain. To gain insight into the function of this domain, structural studies including NMR and SAXS analysis were performed. We find that the MuLV IN NTD is structurally homologous to that of the prototype foamy virus (PFV) and structural comparisons are presented.
For NMR structural studies, the high cost and technical difficulty of producing perdeuterated proteins was initially problematic. By developing and optimizing the cSPP(tet) system, a modified version of the condensed single protein production (cSPP) system, high-quality perdeuterated proteins were obtained at a significantly reduced cost. This approach was critical to defining the dimer interface in the MuLV IN NTD solution structure. Applications of this new technology for use in combinatorial approaches to structure determination are also discussed.
Additional studies focused on the MuLV IN C-terminal domain (CTD). Biochemical analysis of various IN CTD mutations gave rise to a model for transcriptional repression of unintegrated viral DNA mediated by IN. We demonstrate that when combined with mutations in IN, expression from unintegrated viral DNA can be increased in the presence of histone deacetylase (HDAC) inhibitors. Disruption of the IN structure assembled on the viral DNA ends would allow for access of the host transcriptional machinery.