The Funciton Analysis Of Human Bocavirus1Non-structural Protein Np1

Human bocavirus (HBoV1) was identified within pools of human nasopharyngeal aspirates obtained from individuals with respiratory tract illnesses in2005. The alignment analysis indicated that the full-length nucleotides as well as amino acid sequences significantly matched those of bovine parvovirus (BPV) and canine minute virus (MVC), two closely related members of the genus Bocavirus within the subfamily Parvovirinae of the family Parvoviridae, The currently known members of the Bocavirus genus include BPV, MVC, HBoV1and the recently identified porcine bocavirus (PBoV). The HBoV1has been frequently detected worldwide and diagnosed under2-year-old children with upper or lower respiratory tract illness. Subsequently, additional human bocaviruses including HBoV2, HBoV3and HBoV4have been found mainly in human stool specimens. Accumulable evidences demonstrated that infections of HBoVl might be associated not only with respiratory tract illnesses, but also with gastroenteritic diseases.The full length genome of HBoV1that has been sequenced is5.5nts containing two major open reading frames encoding at least an overlapping VP1/VP2capsid protein and a multifunctional nonstructural protein NS1. In contrast to other parvoviruses, Bocaviruses contain a third unique open reading frame named mid-ORF that encodes a NP1protein, which shares47%amino acid identity with NP1of MVC and BPV, but not with any protein of other parvoviruses.1. The nonstructural protein NP1induced cell apoptosis in Hela cellsPrevious study demonstrated that proteins of HBoV1similar to those of MVC failed to induce the cell death in A549cells, which may be considered as a common feature of bocavirus. However, our work revealed that the nonstructural protein NP1of HBoV1could induce apoptotic cell death in human Hela cells in the absence of virus replication and expression of other viral proteins. The apoptosis was confirmed by measaring apoptotic characteristics including morphological changes, DNA fragmentation as well as by flow cytometric analysis, and measurement of caspase3activity. Loss of mitochondrial membrane potential with consequent release of cytochrome c and activation of caspase9led to the increased ratio of Bax/Bcl-2, clearly indicating that HBoV1NP1induced apoptosis in Hela cells through mitochondrion-mediated intrinsic pathway. Moreover, a transient S-phase and G2/M arrest occurred in pNPl-EGFP transfected Hela cells. The analysis of mutants of NP1showed that the N terminal domain may be critical not only for nuclear localization, but also for apoptosis induction. In contrast to the critical role of N-terminal domain in apoptosis, all N-terminal and C-terminal mutated NP1proteins were unable to induce cell cycle arrest, suggesting that the NP1induced cell cycle arrest and apoptosis through alternative mechanisms. 2. Modulation of nonstructural protein NPl on cellular transcriptional factorsRecent studies revealed that NP1bound to the DNA-binding domain of IRF-3, resulting in the interruption of an association between IRF-3and IFNB promoter, which finally blocked IFN-β activation. These results indicate that HBoV1NP1blocks IFN production through a novel mechanism which is the first study to investigate the modulation of innate immunity by HBoV1. B19NS1protein acts as a transcriptional transactivator that regulates a variety of cellular transcription factors, including the expression levels and activities of cellular factors. Thus, NS1may play a critical role in the mechanism of viral pathogenesis in B19infection. In the present study, the modulation of NP1on cellular transcription factors was analyzed by the Dual Luciferase Reporter Assay System and the effect of expression of cytokines TNF-a and IL-6were detected by ELISA and Real-time PCR. The luciferase based mammalian two-hybrid system was used to investigate whether the function of NP1protein resulted from oligomerization. The result showed that NP1activated the transcription factors AP-1, STAT3and STAT1, but not NF-κB in293T cells and that NP1didn’t increase IL-6expression, but may up-regulate TNF-a transcription. Taken together, our data suggested that the function of NP1may not be due to oligomerization.3. Characterization of the complex dual nuclear localization signals of NP1The non-structural protein NP1of HBoV1was found to be a nuclear localized protein and to play important role in DNA replication as well as in the evasion of host innate immunity. The sequence analysis shows that NP1possesses a non-classical nuclear localization signal (ncNLS)(amino acids7KDKHRSY UKRKGSPERGERKRHWQTTHHRSRSRSPIRHSGER47), which is rich in basic amino acids arginine(R), lysine(K),and serine(S), and a classical bipartite nuclear localization signal (amino acids14-28). A nucleolar localization signal (NoLS) of NP1was identified and mapped to amino acids20-40. Cotransfection of the C-terminal deletion mutants of NP1into the Hela cells indicated that The NP1protein was colocalized with a nucleolar marker of B23.1protein. Furthermore, our results indicated that NP1didn’t possess a PY-nuclear localization signal (PY-NLS) verified by transfection of cells with mutants of the critical amino acid clusters. However, the critical amino acids102and105proline (P), particularly amino acid105(P), played a critical role in NP1subcellular localization. Our data reveal that a novel nuclear localization signal is required for nuclear accumulation of HBoV NP1protein and provide some useful information for further understanding its biological function during viral replication.4. Screening of cellular proteins interacting with the NPlThe NP1protein was expressed and purified using the pMAL prokaryotic expression system. A T7phage display cDNA library from human liver cells was screened with the fusion protein MBP-NPl and the selected positive clones were identified by DNA sequence and analyzed using the BLAST program in NCBI. Our results showed that Nup214and NOLC1proteins were potential candidates as novel interacting partners of NPl protein. These results will be confirmed in near future study which would provide important evidences for studying the pathogenesis and mechanism of HBoVl.