He Regulatory Role Of Non-structural Protein On P6Promoter Activity And The Nuclear Transport Mechanism Of Human Parvovirus B19

Human Parvovirus B19(HPV B19), known to be one of the two pathogenic human viruses, belongs to the family Parvoviridae, genus Erythrovirus. As a major pathogen, B19virus causes a range of diseases for children or adults and results in fetal anemia, abortion and fetal hydrops for pregnant woman.Parvovirus B19has been gained more and more attention as the development of technologies and the improvement of people’s disease prevention knowledge. Little progress has been made in the viral replication mechanism and culture in vitro due to the lack of efficient cell model for viral replication. Most studies of B19were mainly focused on molecular epidemiology and diagnosis but not on viral pathogenic mechanism. The aim of the present study was to investigate the effect of the non-structural protein of B19on viral promoter and the intracellular cytokine promoter, and the nuclear transport mechanism of the non-structural protein. These results would provide theoretical basis for the further studies in the effect of non-structural protein of B19on viral infection cycle and viral replication mechanism. The main results of this study are summarized as follows:(1) Regulatory role of non-structural protein of B19on the promoter activity of p6and intracellular cytokineThe p6promoter is the sole functional promoter controlling the transcription of the viral genome and regulating the synthesis of at least12transcripts by alternative splicing and selective polyadenylation. Like other parvoviruses, the p6promoter activity can be regulated by several cellular factors such as Spl/3, YY1and hGABP. Using the transcription factor binding site profile database, we identified the potentially and experimentally-proven cis-acting regulatory DNA elements in p6promoter and predicted three putative ATF/CREB binding sites located at nt.265to343and an E2F consensus element located at nt.386-394. We constructed the EGFP and luciferase reporter gene vectors under the control of the p6promoter, respectively. The data of fluorescent microscopy observation of the EGFP and luciferase reporter assays demonstrated that these predicted ATF/CREB consensus elements influence the p6promoter activity significantly and that the putative E2F binding site does not play any role in p6promoter activity. Meanwhile, p6promoter showed a high activity in different non-permissive cells, but lower than that of CMV promoter. In addition, methylation of the p6promoter by in vitro modification resulted in significant decrease of its activity, indicating that the methylation modification of CpG island in p6promoter significantly inhibits the expression of its downstream genes.The NS1has been found to regulate the promoter activity of viral p6and of several intracellular cytokines, whether the other non-structural proteins possess similar function has not been reported. We did not observe any transactivation of the small non-structural11kDa protein on the p6promoter via luciferase reporter assay, however, we found that, as NS1, the putative protein X of B19transactivated the p6promoter whose DNA elements were located at the first78nts of the N-terminal from nt.265to343containing three putative ATF/CREB binding sites, one EBS element and one Spl/3binding site. This result was in agreement with the pattern that NS1protein transactivates the p6promoter through binding with the ATF/CREB consensus elements.11kDa protein did not upregulate the p6promoter activity, but significantly induced the promoter activity of intracellular TNF-a, IL6STAT3. Our results further confirmed that the11kDa protein upregulating IL6promoter activity was dependent on NF-κB signaling pathways, mainly through degradation of cellular IκBα, induction of p65into the nucleus, and ultimately activated the downstream IL6promoter activity..(2) Cellular localization and nuclear transport mechanisms of non-structural proteins of B19After successfully invading host cells, parvovirus complete its viral genome replication and its viral particle packaging within the nucleus. In order to avoid impact of apoptosis or self defense mechanism of the host cell, parvovirus evolved a priority mechanism in the long-term evolutionary process via nuclear export before completion of cell decline and lysis. Numerous studies have determined that many viral non-structural proteins play roles in their own nucleocytoplasmic transport, viral RNA, viral protein or viral particles export which was mediated by interacting with CRM1. Previous studies have shown that blocking expression of the NS1and11kDa protein significantly reduced B19infectivity and that the deletion of the11kDa protein dramatically affected VP2capsid production and trafficking in infected cells. Therefore, we speculate that non-structural protein NS1, particularly11kDa protein may mediate nuclear export of viral particles.In this study, we explored the cellular localization and nuclear transport mechanisms of non-structural proteins, NS1,11kDa and7.5kDa of B19, and found that NS1and11kDa proteins were exported from nucleus which was dependent on CRM1pathway. Morever, we confirmed that NSI and11kDa protein interacted with CRM1using mammalian two-hybrid method and determined the critical amino acids sites that were responsible for their nuclear export through a series of truncated and point mutations. In addition, the data of subcellular fractions and confocal laser microscopy revealed that the7.5kDa protein was also mainly distributed in mitochondria which was independent on CRM1pathway. Further studies also determined the critical amino acid sites that were responsible for its subcellular localization. Taken together, the results of the role of cellular localization and nuclear export mechanisms played by the non-structural proteins of B19provide some useful information for further study of the viral replication cycle, such as nuclear export process of viral RNA and viral particles as well as for the development of antiviral drug targets.