Molecular analysis of Vaccinia Virus Entry

• Main Authors: Chang, Yuan-Shau, 張淵壽
• Other Authors: Wen Chang
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/47047066108784003879

碩士 === 台北醫學院 === 細胞及分子生物研究所 === 84 === Abstract Vaccinia virus (VV) is an enveloped DNA virus with wide host range andenters cells via plasma membrane fusion. Previous studies have identified amonoclonal antibody(mAb) B2 that recognized cell surface molecules and neutralized 95% VV infection by blocking virus binding. In addition, lambda expression cloning with this mAb resulted in isolation of a 4.2 kb cDNA cloneA1. This A1 cDNA contains an open reading frame (ORF) of 1,300 amino acidsand shares no homology with any known sequences in database. The major objectives of this study are to analyze the gene product of A1 cDNA and to determine if it facilitated VV entry. A1 cDNA was fused with bacteria glutathione-S-transferase (GST) to produce A1-GST fusion protein,which intern was used as an antigen to generate more monoclonal antibodies that could block VV infection. Four mAbs 1-5E-5H mAb, 5-4H-3B mAb,4-6G-5B mAb, and 4-1D-5G mAb were isolated. 1-5E-5H and 5-4H-3B could approximately contribute to 95% of efficiency for blocking the virus infection,while 4-6G-5B and 4-1D-5G mAb could only reach 29% and 50% respecitvely. In order to map out the epitopes within A1 protein for these mAbs, aseries of A1-GST 3'-deletion clones were generated. The result indicated thatthese mAbs recognize four different epitopes localized at the N-terminal regionof A1. In addition, these mAbs also stain cell surface indicating that the recognition sites were situated outside of cells. Therefore, this A1 protein couldbe a type I transmenbrane protein. To understand whether the A1 gene product serves as the cellular receptor of VV, a plasmid expressing the full-length A1 cDNA in mammalian cells was prepared and used to determine the role of A1 cDNA clone in virus entry. Futur study will determine ifoveroxperssion of the A1 gene product will result in increase of virus binding.This study also intend to investigate the role of viral envelope proteins in virus attachment during virus entry. Each envelope proteins were overexpressed in E. coli and their ability to interfere with mAb B2 interactionwith cells was measured. On the other hand, mutant viruses defective of A34R, A36R, B5R, or F13L of envelope proteins were also used to determine the effect of envelope proteins in virus entry. Results from these expermentsrevealed that lack of above envelope protein does not affect the mechanism ofvirus entry.