All 4 di-leucine motifs in the first hydrophobic domain of the E5 oncoprotein of human papillomavirus type 16 are essential for surface MHC class I downregulation activity and E5 endomembrane localization.
The E5 oncoprotein of human papillomavirus type 16 downregulates surface MHC Class I and interacts with the heavy chain of the MHC complex via the first hydrophobic domain, believed to form the first helical transmembrane region (TM1) of E5. TM1 contains 4 equally spaced di-leucine (LL1-LL4) motifs. Di-leucine motifs have been implicated in protein-protein interactions and as localization signals. To see if any of the 4 di-leucine motifs of TM1 are involved in MHC downregulation by E5, we mutated each LL pair into valine pairs (VV1-VV4), as mutation of leucine to valine is not expected to cause major structural alterations in E5. We found that all 4 mutations disrupted the intracellular location of E5 and abrogated its MHC I downregulating activity; however VV2 and VV4 mutants were still able to interact physically with the MHC I heavy chain (HC) in vitro, while VV1 and VV3 mutants had lost this activity. We conclude that LL1 and LL3 are necessary for the interaction with HC, but LL2 and LL4 are not. However all 4 LL motifs are responsible for the proper localization of E5 in the Golgi/ER, and the displacement of E5 from this location contributes to the abrogation of MHC I downregulation. LL1 and LL3 motifs are expected to be on one face of the TM1 helix and LL2 and LL4 on the opposite face. We propose that E5 interacts with HC via LL1 and LL3 and that all 4 di-leucine motifs act as a targeting signal.
Cortese
Marc S
MS
Division of Pathological Sciences, Institute of Comparative Medicine, University of Glasgow, Glasgow, Scotland, UK.
Ashrafi
G Hossein
GH
Campo
M Saveria
MS
D020816:Amino Acid Motifs; D002478:Cells, Cultured; D015536:Down-Regulation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D052162:Human papillomavirus 16 / Q000502:physiology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism; D007930:Leucine / Q000737:chemistry*; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D000086082:Viroporin Proteins
D015395:Histocompatibility Antigens Class I; D007930:Leucine; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; C054859:oncogene protein E5, Human papillomavirus type 16; D000086082:Viroporin Proteins