The Effect of Single Amino Acid Mutation on the Envelope Protein of Japanese Encephalitis Viruses Binding to Glycosaminoglycans Distributed on the Surface of Host Cells

• Main Authors: Liu, Hsuan, 劉璇
• Other Authors: Chen, Wei-June
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/33567174251996142266

碩士 === 國立臺灣大學 === 流行病學研究所 === 91 === Japanese encephalitis (JE) is one of important mosquito-borne viral diseases in Asia. Japanese encephalitis virus (JEV) is able to replicate in cells derived from either mosquitoes or mammals. It has been known that the viral envelope (E) protein interacts with molecule(s) on the cell membrane as soon as JEV infects cells. Heparan sulfate (HS), a member of highly sulfated glycosaminoglycans (GAGs), distributed on the cell surface has been postulated to play a role in JEV infection. However, the molecular mechanism of JEV infection was still not clear thus far. In this study, two sets of virus variants derived from two virus strains were chosen for most experiments. The set of TL4/TS1 was from T1P1 strain isolated form Armigers subalbatus in 1997; while the set of CJNL1/CJNS1 was from CJN strain isolated from the brain of the encephalitis patient in 1998. Each set of virus strains possessed single amino acid variation on the E protein between two variants; variation of Glu/Lys on E-306 was for TL4/TS1 and the same variation on E-138 was for CJNL1/CJNS1. To evaluate the role of GAGs on the process of virus infection, heparin (a close structural homologue of HS) and a GAG-deficient cell line were utilized to observe the differences of plaque formation derived from target cells that were infected, attached, or penetrated by JEV. The relationships of JEV mutation, especially on the residue 306 or 138 of E protein (E-306 or E-138), with HS were also evaluated in this study. Besides, we utilized anti-HS monoclonal antibody to detect the abundance of HS on the membrane of different cells and then to elucidate its interaction with the virus infection to. In this study, the term of ED50 (50% effective dose) was defined as the heparin concentration that inhibited formation of 50% plaque number when the virus, in a mixture with heparin, was inoculated to target cells. The ED50 of TL4/TS1 infection (attachment and penetration) to cells were 17.3/5.2 U/ml (12.6/13.1 U/ml and ＞250/13.2 U/ml). On the other hand, the ED50 of CJNL1/CJNS1 infection (attachment and penetration) to cells were 56.8/5.4 U/ml (59.0/5.7 U/ml and ＞250/11.6 U/ml). In addition, heparin was shown differentially effective on the plaque formation during JEV infection (including attachment and penetration) to host cells. This phenomenon was confirmed by distinct efficiencies of JEV binding to the wild-type CHO cell line and its mutant that defects in GAG biosynthesis. Variations on either E-306 or E-138 from Glu to Lys have been demonstrated to increase the capability of virus binding to heparin/HS. For target cells, however, E-306 variation affected the virus penetration only while E-138 variation affected both attachment and penetration of the virus. It indicates that both variations were important for HS-binding. As a result, JEV may utilize HS as the receptor to attach and then to penetrate host cells. However, HS was probably not the only receptor for virus penetration. Other molecules such as proteins on the cell surface may also be involved either alone or in accompany with HS to determinate virus penetration. In the assay of HS abundance on different cells using anti-HS monoclonal antibody, it revealed that the more susceptible to JEV the cells the higher the HS on the cell membrane. It implicated that the infectivity of JEV to mammalian cells highly depended on the abundance of HS distributed on the cell surface. However, it seems not true for mosquito cells as relatively low abundance of HS has been found on their cell surface. Apparently, the pathway of JEV infection to mosquito cells was rather different from that to mammalian cells. In conclusion, JEV was necessary to utilize HS on the process of infection, including attachment and penetration, to mammalian cells. Both E-138 and E-306 of JEV were extremely important for HS-binding although it remains to be worked out in actual effects of these variations on virus entry into host cells, especially the process of attachment and penetration. The results also indicated that tissue tropism of JEV may be derived from the abundance of HS on the surface of mammalian cells. However, the infection route of JEV to mosquito cells was apparently different. In the future study, it will be necessary to define more precisely the role of variations E-306 and E-138 in the virus entry. In addition other receptors on the cells that are possibly involved in the virus are also expected to be identified.