| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 93 === Subgenomic replicons of positive-stranded RNA viruses have been shown to be valuable tools for studying viral replication independently of virion assembly, screening inhibitors, expressing heterologous genes, and developing new immunization vectors. Recently, replicons of several flaviviruses have been successfully constructed, including Kunjin viruses, tick-borne encephalitis viruses, West Nile viruses, yellow fever viruses, and dengue viruses（DENV）. Although replicon of dengue virus type 2（DENV2）was reported recently, an efficient encapsidation system for dengue replicon has not been developed yet. The first specific aim of this study is to establish stable clones that produce subviral particles containing the precursor membrane and envelope （prM/E）proteins of DENV. The second aim is to establish replicons of dengue virus type 4（DENV4）and DENV2. The third aim is to study the encapsidation of replicon into virus-like particles （VLPs）by using appropriate packaging cell line.
Stable cell lines constitutively expressing prM and E proteins of DENV1, DENV2 and DENV4 were successfully established to produce subviral particles efficiently. Immunofluorescence analysis revealed high levels of expression of E protein except the DENV1 prM/E stable clone. In the pellets derived from ultracentrifugation of supernatants, expression of prM and E proteins was shown by Western blotting analysis, indicating that these prM/E stable clones were capable of producing subviral particles.
Both replicons of DENV4 and DENV2 were constructed. One type of replicons, designated as T7C101E470NSpFast（DENV4）and SP6C101E470NSp2（DENV2）, lacks the entire prM and the majority of E genes. Another type of replicon designated as T7C30E470NSpFast（DENV4）, lacks the entire prM and the majority of C and E genes. Immunofluorescence analysis revealed that these three replicons were able to replicate and express proteins in BHK cells and 293T cells, though the efficiency is not high especially in 293T cells.
To investigate whether prM/E stable clones can package DENV replicons, the in vitro-transcribed replicon RNA were transfected to prM/E stable clones（ pCDJssD2J396 and pCDJssD4J396 ）, as well as to and 293T cells transiently transfected with a construct that express prM/E of DENV2（pCBDssD2）. Four days after RNA transfection , VLPs derived from culture supernatants were inoculated to fresh BHK cells to examine the expression of NS1 protein by immunofluorescence. Only the DENV4 replicon, T7C101E470NSpFast, can produce infectious VLPs after its transfection to prM/E stable clone pCDJssD2J396. No infectious VLPs was produced in the pCDJssD4J396 stable clones and cells transfected with pCBDssD2. This could be due to low efficiency of transfection of RNA replicon, since expression of NS1 protein by T7C101E470NSpFast replicon in pCDJssD2J396 stable clone was better than that in other two prM/E expressing cells. Take together, these results indicated that dengue prM/E stable clone, pCDJssD2J396, can be used as a packaging cell line for DENV replicon to generate infectious VLPs for future study. However, the ability to package replicon for other prM/E expressing cells needs to be further studied.
The overall objective of this study is to establish an efficient and convenient packaging system for DENV replicon. The packaging system can be used to study the mechanism of the encapsidation step of DENV replication. The infectious VLPs derived from the system can be used to study the mechanism of DENV entry and to screen entry inhibitors as well.
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