Assemblies of ORF8a from SARS-CoV, and BST-2 with Vpu from HIV-1 within the lipid membrane

• Main Authors: Cheng-Chang Chen, 陳政彰
• Other Authors: Wolfgang B. Fischer
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/eqg344

碩士 === 國立陽明大學 === 生醫光電工程研究所 === 97 === 　　Many genomes of viruses encode small membrane spanning proteins which are proposed to alter membrane permeability or cell response. These membrane proteins are getting into the focus for antiviral therapy since they are essential for some of the viruses. One of the common themes of the mechanism of function of the proteins is to self-assemble to form the functional form. 　　In the first part of this study, the open reading frame (ORF) 8a membrane protein encoded in structural region of Human Severe Acute Respiratory Syndrome Coronavirus is investigated. The full length ORF8a protein is 39 residues long and contains a single transmembrane domain. Full length protein is synthesized using solid phase peptide synthesis. When reconstituted into artificial lipid bilayers it forms cation-selective ion channels. The bilayer recordings show ion channel activity with a main conductance level of around 8.5 pS also at elevated temperatures (38.5°C). In silico studies with a 22 amino acid transmembrane (TM) domain are done to assess conformational space of the monomeric ORF8a helix. With this monomeric helix homooligomeric helical bundle models are built and embedded in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycerol-3 -phosphatidylcholine bilayer. Results of computational modeling suggest that SARS ORF8a could form a pentameric pore. 　　The second part of this study focuses on the evaluation of amino acids within the TM domains (TMDs) of Vpu and BST-2 and their role in the putative assembly process. The human and non-human primates BST-2 TMDs, including the LG-insertion mutant, are compared. Data from flow cytometry assays of Vpu-mediated modulation of BST-2 are presented. The data illustrate that the L29G30 TMD mutant of Rhesus BST-2 is down regulated from the cell surface by Vpu and also expressed at a lower level than other BST2 types. These results imply that an interaction between TMDs of BST-2 and Vpu should exist. We also have started to model the interaction of the TMDs of BST-2 and Vpu mutant (A18H) using computational methods.