Studies On Modification, Modulation Of Tobacco Mosaic Virus And Its Interaction With Antofine Analogs

Research on tobacco mosaic virus (TMV) has played a leading role in the development of virology for more than a century. TMV is one of the most harmful plant virus found worldwide, meanwhile, also the most deeply studied on the configuration, structure, chemical composition, assembly, subunit, DNA replication, protein translation, and cell to cell movement, et al. TMV exhibits most of its popularity as a model virus to the investigation on regular assembly mode and nano-dimension character. Studies on TMV have been last for a long period, and its prominent position in both fundamental and applied research will still continue to occupy. Our study interests focused on two major aspects:one, prevention and inhibition of TMV, which is a hot subject with practical significance in the field of research and development of new pesticides. Based on our previous work, we wondered the exact interaction model between TMV and Antofine; another, regulation of TMV. We hope to increase the understanding of the character and application of TMV, by means of its modification and length control.This article is mainly focus on the following four parts:First:Antofine is reported to have shown remarkable inhibition on TMV, and its analogs have pronounced activities including cytotoxicity, antitumor activity et al, which attracts many attentions. Our group has reported that high order of molecular recognition to bulged hairpin DNA afforded by Antofine, also got some deduction on Antofine’s basic mode of its interaction with DNA by means of fluorescence and UV-Vis spectroscopy. On the basis of these work, herein, we designed and synthesized a series of bulged DNA containing much more subtle differences in comparison with HT3AGT which has the best affinity with Antofine in previous study. We ascertained the exact interaction mode between Antofine and bulged hairpin DNA.Antofine is a well-known inhibitor for TMV, but its target is still unknown. Dr. Ruoyu Zhang has found the strong affinity between Antofine and TMV RNA. Herein, we determined the Kd value of Antofine and TMV CP by equilibrium dialysis. Combined with the determination of affinity with TMV RNA and assembly inhibition ability of Antofine analogs, we found that the activities of antofine and its analogs to inhibit TMV assembly may correlate with their binding affinity to TMV RNA. So we deduced antofine analogs inhibit TMV assembly mostly by virtue of interacting with TMV RNA.Second:OriRNA plays a dominate role in TMV lifecycle and total assembly inhibition, thus, our group using SELEX, constructed a library of-109 different RNA sequences with 16 random sites. We sequenced all the members in the final library after 7 cycles’selection, and conclusions could be drawn from the analysis of the mutations:results from the statistical point of view, it is not obvious of the feature that the apex contains the specific trinucleotide ’NNG’, but N prefer A and G, while C is dislike; G in the second ’NNG’ of the apex maybe important to the assembly ability; the melting ability of the base pair closed to the apex is crucial to the assembly; G-U wobble pair may play a certain role in the recognition between OriRNA and TMV CP. From the view of the abundance of sequence, we found that wild-type OriRNA has the maximum frequency of its appearance, which indicated that both artificial evolution and natural selection got the same result.Third:TMV has a hollow cylindrical structure with a high aspect ratio and formed through the periodical self-assembly of the TMV CP and TMV RNA. Because of its well-characterized nanosacle structure, TMV has been utilized as a template for functionalization with inorganic and organic molecules both in the inner cavity and on the exterior surface. TMV has also been used in the biotechnological sphere:as the source of transgenic sequences conferring virus resistance, in vaccines consisting of TMV particles genetically and in systems for expressing foreign genes. In order to better understanding these applications of TMV, we need to get much more information on its assembly mechanism while the character of TMV surface. We described the application of photochemical crosslinking of coat protein of TMV particle and its effect on infectivity and stability of TMV. Compared to native TMV, the crosslinked TMV showed lower thermostability and resistance to dilute alkaline, though it remains physical integrity; crosslinked TMV nearly lost its infectivity on tobacco leaves. On the other hand, Alkylsulfonium salts (ASS) have been reported as powerful alkylation agents. We use a tandem method of sulfonium alkylation and "click" chemistry (CuAAC) for TMV modification. This facile modification should be useful in bionanoscience.Although TMV has such promising features for its application to nanoscale materials, the length of wild-type TMV rod which is limited to 300nm restricts its further application. We may replace the rod structure by other different lengths of RNA or DNA using suitable methods to realize length control and functional reconstruction. We adopted two methods as follow:first, we wondered whether the OriRNA can lead the assembly of DNA. If it works, we can be free to control the rod length, so we constructed a 153nt RNA-DNA hybrid in which the RNA part comprised the OriRNA of TMV, then determined the assembly ability of this hybrid; second, using PCR and transcription in vitro to construct different lengths of TMV RNA strands, during assembly procedure with TMV CP, we may get different lengths of virus rod evenly, to realize precise controlling of nano-scale particles of TMV.Finally:people try many methods to achieve the modification of TMV, but neither by enzyme nor by chemistry can realize site-directed modification. So we considered to express TMV coat protein in E.coli, then assemble reconstituted or modified CP with TMV RNA into particles. Herein we successfully expressed the TMV CP in E. coli and checked its assembly ability at the same time.Research on the interaction mode of Antofine and tobacco mosaic virus has opened up our thinking about the development of new antiviral strategies. Modification of TMV surface has deepened our understanding of its properties. Precise control of nano-scale particles of TMV has laid the foundation for TMV as a drug carrier. In vitro expression of TMV CP has enriched our means to control it. Virus infection is a severe public health problem related to serious personal, social, and economical consequences, herein, we ascertained the target of Antofine, developed some methods to modify TMV surface, which can be beneficial to the anti-viral research and application of TMV.