Characterizing the binding of HSV-1 VP16 to the cytoplasmic tail of glycoprotein H
Gross, Sarah T.
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The Herpes Simplex Virus type 1 (HSV-1) particle contains the viral genome enclosed by a proteinaceous capsid, which is surrounded by protein layer known as the tegument, and enveloped by a host-derived membrane that is studded with viral glycoproteins. During HSV-1 envelopment, capsids, tegument polypeptides and membrane proteins assemble at the site of budding and a cellular lipid bilayer becomes refashioned into a spherical envelope. Though the molecular interactions driving these events are poorly understood, several lines of evidence suggest that associations between envelope protein cytoplasmic tails and tegument polypeptides may play important roles in this process. In support of this hypothesis, my thesis research has focused on characterizing the interaction between the cytoplasmic tail of the viral glycoprotein gH, to tegument protein VP16.;Using a GST-fusion binding assay, we found that the C-terminal cytoplasmic tail of gH interacts with the tegument protein, VP16, in a temperature dependent manner, binding only at 37°C but not at lower temperatures. Using in vitro translation of VP16, we demonstrated that VP16 binds to the gH tail independently of other viral factors. By performing alanine scanning mutagenesis, we identified the binding site of VP16 on gH, and found that mutation of a proline residue at the center of the gH tail enhances binding of VP16 to the gH C-terminus, and partially relieves the temperature dependence of VP16 binding.;To determine whether these results are significant in vivo, we performed coimmunoprecipitation experiments using HSV infected cells and cells that have been both infected and transfected with a GFP-gH tail fusion construct. These experiments have shown that both wild type and mutant gH tails interact with VP16 in vivo with similar characteristics to in vitro binding.;The temperature dependence of the VP16/gH interaction prompted the formation of a collaboration with Drs. Mark Girvin and Douglas Kamen to study the structure of the gH tail peptide and the P → A mutant peptide using NMR. These studies have shown that the WT gH tail is unstructured at binding-permissive temperatures and is structured at lower temperatures where VP16 cannot bind gH; the P → A mutant is unstructured even at lower temperatures.