Characterization of the Herpes Simplex Virus 1 VP11/12-tyrosine based binding motifs for Src family kinases, p85, Grb2 and Shc
Infection with HSV-1 triggers several events specifically designed to manipulate cell signal transduction pathways. Two major signaling cascades targeted by the virus are the PI3K/Akt-pathway and the TCR-signaling pathway. Prior to the studies presented in this thesis, it was known that the tegument protein VP11/12 associates with cellular proteins that are directly involved in those pathways. Specially, it was suggested that VP11/12 associates with SFKs, p85, Grb2 and Shc through specific
... ine-based binding motifs that are located within the C-terminal region of VP11/12. Here, we first generated point-mutated viruses with inactive binding motifs and assessed the ability of mutant VP11/12 to associate with the SFKs, p85, Grb2 and Shc. I found that inactivation of the predicted binding motifs for SFKs, Grb2 and Shc eliminated the protein-VP11/12 associations. In the case of p85, I was able to demonstrate that inactivation of the predicted p85-binding motif significantly reduced, but did not eliminate, the VP11/12-p85 interaction. From these results, it became evident that VP11/12 requires the phosphorylation of specific tyrosine-based binding motifs within its Cterminal region in order to associate with SFKs, p85, Grb2 and Shc. Next, I determined the downstream effects of eliminating these protein-protein associations. I provide data suggesting that VP11/12 requires the recruitment and activation of SFKs to further associate with Grb2, Shc and p85 as well as to induce global phosphorylation of VP11/12. Further, I was able to demonstrate that VP11/12 must associate with SFKs, Grb2 and p85 in order to activate Akt during infection. In addition, I provide data suggesting that VP11/12 needs to associate with SFKs in order to severely reduce TCR signaling events upon transfection. Taken together, this data fully supports the Wagner-Smiley model demonstrating that VP11/12 mimics an activated receptor in that VP11/12 associates with cellular proteins that are directly involved in the stimulation of the PI3K/Akt-as well as the TCR-signaling pathways. First I would like to thank Dr. Jim Smiley for serving as my supervisor and providing an excellent training environment. I would also like to express my thanks to all members of the Smiley lab, especially Holly Bandi, Dr.