Identification and characterization of a small modular domain in the herpes simplex virus host shutoff protein sufficient for interaction with VP16.
The herpes simplex virus transactivator VP16 and the virion host shutoff protein vhs are viral structural components that direct the activation of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection. Recent studies show that VP16 and vhs physically interact with each other in vitro and in infected cells, suggesting that their respective regulatory functions are coupled. In this report, we used the yeast two-hybrid system and affinity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16. Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residues 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing complex formation with VP16 in vivo and in vitro when fused to a heterologous protein. Site-directed mutagenesis of this region identified tryptophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable complex formation in vitro. These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16.
Schmelter
J
J
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Knez
J
J
Smiley
J R
JR
Capone
J P
JP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012260:Ribonucleases; D012441:Saccharomyces cerevisiae / Q000235:genetics; D018139:Simplexvirus / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C070722:virion host shutoff protein, Simplexvirus; D014364:Tryptophan; D012260:Ribonucleases