| The canonical RNAi pathway has been characterized most extensively in D. melanogaster, in which it was found that long double-stranded RNA(ds RNA) is diced progressively from the termini into a pool of 21-nucleotide si RNA duplexes by the ds RNA-specific endoribonuclease(RNase) Dicer. These si RNAs then select and destroy their m RNA targets by base-pairing with and guiding the endoribonucleic cleavage(or slicing) of the target m RNA by an Argonaute(AGO) protein in the middle of the si RNA–m RNA duplex.As an old gene regulation mechanism, RNA interference(RNAi), plays an important role in regulating development, as well as responding to external environment pressure. Due to its power in gene silence, RNAi has also been well known to be the major antiviral immune mechanism in plants and insects. As a general dogma in nature, plant virus and insect viruses have evolved the special mechanism by encoding viral suppressors of RNA silencing(termed VSR) that can effectively inhibit the RNA-based virus immunity, thus facilitating virus multiplication and transmission in plants and insects.At present, albeit RNAi pathway has been verified in mammals, and it remains questionable whether RNAi can be utlized by mammals to combat against the invading viruses. RNAi seems a little naive and primal for mammals because that mammals have evolved more advanded and powerful antiviral immunity system including interferon.Japanese encephalitis virus(JEV) belongs to the flavivirus genus, and it is a kind of arboviruses spreading by Culex pipiens. Japanese encephalitis virus causes severe encephalitis symptoms and diseases of the nervous system in clinical, and pose a serious threat to human and public health. Its genome is a 10976 bp, single-stranded positivesense RNA molecule. The genome of JEV is a single open reading frame which is flanked by viral untranslated regions. The viral ORF encodes three structural proteins:the capsid protein, membrane protein precursor/membrane proteins, and envelope glycoprotein, as well as seven nonstructural proteins(NS1, NS2 A, NS2 B, NS4 A, NS4 B, NS3, and NS5). As an arbovirus, the natural host of Japanese encephalitis virus includes both insects and mammals, so it is an ideal lagent for the research of RNAi immune pathway. Interferon immune pathway of Japanese encephalitis virus in mammalian hosts has made great progress, and a large number of research results show that the nonstructural proteins of Japanese encephalitis virus(NS4B, NS5) could significantly inhibit the IFN pathway; however, the study of RNAi immune against Japanese encephalitis virus in insect or mammal is still in the initial stage. Finding and confirming the VSR of virus has an important significance to understand the molecular mechanism of virus infection, and also can be used as a tool to prove the existence of the immune mechanism of RNAi in mammals.In this study, the RNAi antagonizing functions of JEV protein were analyzed in-depth through the eukaryotic expression system and biochemical means in vitro. We find that NS2 A can antagonize the RNAi; further, through bioinformatics and reverse genetics technology, we analyzed the relative site on NS2 A protein which may have interaction with ds RNA, and constructed the mutant viruses, of which the biological characteristics were evaluated preliminarily.The content of this study includes the following two parts: 1. Screening VSR of Japanese encephalitis virus and determining its functionB2 protein of Flock House Virus is the well-known VSR. m RNA of EGFP could be regulated by RNAi when transfecting with the ds RNA/si RNAs of EGFP in insect cell. On this part, we transfect the 10 proteins of Japanese encephalitis virus with the EGFP m RNA in the system, and it is identified that NS2 A of Japanese encephalitis virus could protect EGFP m RNA from degradation of RNAi pathway like VSR-FB2. Then,through knocking out the key role of protein in RNAi pathway, Northern blot is choosed to prove that NS2 A of Japanese encephalitis virus has the interaction with ds RNA/si RNAs to function of VSR, and it is determined that NS2 A is the VSR of Japanese encephalitis virus. Since the NS2 A protein may bind to ds RNA/si RNAs and RNA is negativelycharged. The positively-charged amino acids of NS2 A protein may play a key role as a VSR. Then, we compare the different sequence of flavivirus NS2 A protein through bioinformatics methods and find that most of positively charged amino acids are very conservative, and existing the accumulation phenomenon of multiple positive charged amino acids in a certain area. So the positively charged amino acids is likely to be important sites. Further research by I-TASSER protein prediction analysis of Web data, infer the possible folding pattern of NS2 A protein,and the Py MOL software is used to analyze protein surface sites.It is found that the positively-charged amino acids sites are in the folding connecting region of transmembrane protein. Therefore, we infer K26, T33, K84, R98, R140,R163, R171, K193, K226 are the key amino acids which can affect the function of NS2 A as a VSR. 2. Construction the VSR deficient mutant virus of Japanese encephalitis virus and analysis of their biological characteristicsFrom the results above, based on infectious clone of Japanese encephalitis virus-E70, we use fusion PCR to construct the T33 I, K84 A, R98 A, R140 A, R163 A, R171 A, K193 A and K226 A,a total of 8 point mutation plasmids successfully. And then through in vitro transcription and transfection of BHK-21 cell line, we have rescued the T33 I, K84 A, R98 A,R140A, R163 A point mutant virus, whereas the R171 A, K193 A and K226 A are invariable. By using reverse transcription, PCR test, virus plaque assay, and indirect immunofluoresce assay, it is determined that the mutant viruses are infectious. Plaque diameters of the T33 I, K84 A, R163 A mutant viruses are significantly less than the wild type virus E70. Subsequently, E70 and mutant viruses are inoculated in C6/36, Aag2 and 293 T cell lines to show replication capacity of virus on cell. The results illustrate that: the replication capacity of R98 A, R140 A and R163 A are basically as same as E70 in different cell lines; the replication capacity of K 84 A are significantly reduced in the three cell lines tested, indicating that K84 A may affect the replication of virus genome or virion assembley; the replication capacity of T33 I is consistent with that of E70 in C6/36 cell lines in which the RNAi pathway is defective,while, it is significantly reduced in Aag2 and 293 T cells whose RNAi pathway is active, and it is indicated that T33 I may affect the function of VSR of Japanese encephalitis. In animal experiments, we measure the neurovirulence and neuroinvasiveness of mutant viruses in neonatal rats and mices. It is showed that when compared with the E70, the neurovirulence of T33 I, K84 A, R98 A are significantly decreased and the neuroinvasiveness of which are relatively weaker.Through the research, we have found and confirmed that NS2 A is one of the important VSRs of Japanese encephalitis virus, and T33, K84 and R98 of NS2 A have an important influence on viral pathogenicity,and these are key virulence(neurovirulence or neuroinvasiveness) sites. The research confirme the key role of RNAi in viral replication in mammalian cells for the first time, and it is very significant to understand the interaction between virus and host. |
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