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UBC Theses and Dissertations

Functional characterization of a small nonstructural protein of the parvovirus B19 Fan, Mannie Man Yee


The pathogenic human parvovirus B19 produces a class of small, 11kDa proteins whose function is as yet undetermined. In the absence of a continuous permissive cell line in which B19 can be readily propagated, in vitro studies have been used to assess the functional role of the 11kDa proteins. In previous studies, the 11kDa proteins have not been shown to be targets of phosphorylation or binding partners for other viral proteins, nor have they demonstrated Zn++-binding or transactivation activities. However, there is preliminary evidence that the 11kDa proteins are capable of interacting with the cellular growth factor receptor-binding protein 2 (Grb2) in vitro, and this thesis aims to investigate this interaction in greater detail. In studies reported in this thesis, far western experiments confirm in vitro interactions between Grb2 and the 11kDa proteins. Moreover, a fusion construct of the 11kDa protein with glutathione S-transferase (GST) is able to specifically pull down endogenous Grb2 from HEK 293 cell lysates, providing further support for our hypothesis that the two proteins interact. Grb2 is an important adaptor molecule in the MAPK signal transduction pathway leading to defined cellular responses. The protein is composed of a series of modular domains each specialized for a different function, including two SH3 domains that are capable of binding proline-rich sequences. Such sequences correspond to consensus "ligand" motifs found in binding partners for Grb2 such as son of sevenless (Sos), a guanine nucleotide exchange factor for the Ras small GTP-binding protein that is implicated in MAPK activation. Sos-like proline-rich regions are also present in the 11kDa proteins. Further studies have focused on the requirement for these regions in the Grb2/11kDa interaction. Far western studies employing 11kDa proteins mutated at proline residues suggest reduced interactions with Grb2. Furthermore, GST-11 mutant constructs do not precipitate Grb2 from mammalian cell lysates as readily as does the wild-type. These results indicate possible involvement of the proline-rich region in mediating interactions of the 11kDa proteins with Grb2, in an SH3- dependent manner. The findings from this thesis strongly support an interaction of the 11kDa proteins with the host protein Grb2 to influence host cell environment during the viral replicative cycle.

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