| Enterovirus71(EV71) belongs to the genus Enterovirus within the family Picornaviridae. Infection of EV71mainly leads to the Hand-Foot-Mouth Disease (HFMD), and severe central nervous system syndrome, which is the main cause of death cases. Currently there are no vaccines of specific antiviral drugs available.The genome of EV71is a single-stranded, positive sense RNA molecule containing a single open reading frame, which encodes four structural proteins (VP1VP2, VP3, VP4) and seven non-structural proteins (2A,2B,2C,3A,3B,3C and3D). The5'and3'end of its genome are untranslated regions (UTR), which contain an internal ribosome entry site (IRES) and other RNA elements. Non-structural protein3C protease (3Cpro) is responsible for the processing of virus polyprotein precursors, and it cleaves some host proteins as well. Thus, it is considered as a key protein in the virus-host interaction. The mechanism of expression and transportation of3Cpro is not clear, therefore, the dynamic time-space character of3Cpro needs to be revealed for the research of EV71pathogenesis.In the present study, the infectious full-length cDNA clone of EV71pandemic strain was constructed, and the recovered virus was identified.3Cpro mutant virus bearing at CCPGCC peptide sequence in its3Cpro was constructed and characterized. The tetracysteine (TC) biarsenical labeling system was further applied to obtain the real-time imaging for the intracellular expression and translocation of3Cpro in host cells. The results paved way for the research of replication and virus-host interaction mechanisms of EV71and other enterovirus.1. Construction and identification of EV71infectious full-length cDNA cloneBased on the pandemic strain, infectious full-length cDNA clone of EV71genome was constructed. The full-length viral genome was amplified by one-step RT-PCR using high-fidelity DNA polymerase, with introduction of SP6promoter and the SnaBI monoclonal site to the5'upstream and Mlu I site to3'downstream of the genome, respectively. This full-length genome was cloned into pEV vector derived from pBR322plasmid. Enzyme digestion and joint-region sequencing were conducted to confirm the construction of infectious full-length cDNA clone.To identify the viability of the recovered virus, the clone was linearized as the template for in vitro transcription. RNA transcript was transfected into RD cells via electroporation or lipofectamin transfection. At48hours after transfection, the cells were harvested and seeded onto RD cells. CPE was observed12h post infection of RD cells, which time was the same as the wild-type virus. RT-PCR results suggested sequence identity between the rescued virus and wild-type virus. Indirect immunofluorescence results showed that the virus-specific proteins can be detected from rescued virus infected cells after infection for6h. Western blot (WB) experiments demonstrated that the virus-specific capsid protein was expressed in rescued virus. There is no difference between the rescued virus and wild-type virus in plaque formation, morphology and emerging time. One step growth curve showed that rescued virus titer reached the highest value of107PFU/ml after48h, indicating the consistent of virus infection and cellular proliferation with the rescued and wild-type virus. In addition, the efficiency of electroporation and lipofectamin transfection for the virus recovery was compared. CPE can be observed48h post electro-transfection under850V and reached100%after96h. However, the CPE can be observed after12h with lipofectamin transfection using the same amount of RNA, and reached100%after48h, suggesting a higher transfection efficiency.All these results above suggested the successful construction of EV71infectious full-length cDNA clone, which was designated as A12, and subsequent genetic modification work can be carried out. Lipofectamin transfection is preferred for the virus rescue, with its advantages of easy operation and high transfection efficiency.2. Construction of EV71genetically engineered virus bearing a TC tagIn order to investigate the expression and transportation of3Cpro using EV71infectious full-length cDNA clone, polypeptide sequence CCPGCC was inserted between the S and E located at181and182sites in3Cpro, which led to the visualization of the reporter virus within the cells. RNA transcribed from linearlized plasmid containing genome of the recombinant reporter virus was transfected into RD cells. The typical EV71CPE was observed12h after transfection, and reached100%after36h; CPE was observed after inoculating of tagged virus and wild-type virus onto RD and Vero cells. After inoculation for rescued virus, the expressions of capsid protein VP1and3Cpro were detected by both indirect immunofluorescence assay and Western blot, and the expression level was progressively and significantly increased in a time-dependent manner. In RD cells, the plaque formed by reporter virus was consistent with the wild type in both morphology and the occurrence time. One step growth curve analysis indicated that the proliferation of the reporter virus was the same as wild-type virus. After continuous passage, the sequence of the passaged virus was the same as parental virus, and the sequence of virus genome was stable with no mutations observed.The chemical imaging of3Cpro localization after virus infection obtained by dual arsenic chromogenic reagent (FlAsH) was in accord with result via the traditional indirect immunofluorescence. Real-time imaging during the virus infection suggested the3Cpro was expressed at4.5h to5h post infection and imported into the nucleus at5h to6h post infection. The results above made clear of the3Cpro nuclear import time after its expression, laying a good foundation to investigate the3Cpro performance, virus replication, and the virus-host interaction mechanism.The above results indicated that we successfully rescued the TC tagged virus. The biological characters of recovered virus was the same as the wild-type virus, and the genome of the tagged virus was stable, in which the inserted TC tag can be stably passaged.3. Real-time analysis of3Cpro using engineered TC tag virusIn order to study the expression and translocation of3Cpro in infected cells with the TC tagged virus, the biarsenical FIASH-EDT2reagent was applied for the TC tag imaging. Results showed that the3Cpro of the TC tagged virus can be fluorescently imaged, which location is the same with traditional IFA. When imaging the whole infection process with the TC tagged virus, it was observed that the expression of3Cpro was set off between4.5h-5h, and the nuclear importing was observed between5h-6h post infection. These results proved the nuclear importing of3Cpro, and clarified the exact time point between its expression and nuclear importing, which paved way for the study of3Cpro function and the research of virus-host interaction. The co-localization of3Cpro and cellular protein CstF-64was carried out, and proved the cleavage of CstF-64with3Cpro. These results suggested that the TC tagged virus is suitable for the research of virus-host interaction.This study has successfully constructed the EV71infectious full-length cDNA clone based on the domestic epidemic strain. Moreover, the virus genome was further modified, and reporter virus was first conducted by biarsenical tetracysteine (TC) technology. The virus was characterized and virus-host interaction was studied with3Cpro. The results confirmed that the181S-182E of C terminal of the3Cpro can bear insertions, which can be used as the potential site for protein marker; the biological characters of TC tagged virus are the same as wild-type virus, and the genome is stable;3Cpro synthesis was set off at early stage of viral infection;3Cpro began to gather and enter the nuclear within1h after synthesized; the TC tagged virus is suitable for the research of virus-host interactions. These findings paved way for the clarification of3Cpro function in virus infection.
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