The Nuclear Export Mechanism Of The Nonstructural Protein NS1of Human Parvovirus B19

Human Parvovirus B19, first discovered in1974, is the one of the two members of the family Parvoviridae which can infect human beings. B19is widespread and causes different symptoms due to different immunologic and hematologic conditions of the hosts infected. The main diseases caused by this virus are erythema infectiosum in healthy immune-competent children and acute symmetric polyarthropathy in adults. As to pregnant women infected by B19lead to fetal death, hydrops fetalis or development of congenital anemia. At present, two major detection methods of B19infection are antigen enzyme-linked immune-sorbent assay (ELISA) and detection of viral DNA by direct hybridization or PCR. B19-specific IgG can efficiently alleviate the gravity of virus infection.The viral particle of B19is a non-enveloped icosahedron embracing a single-strand DNA genome which contains5.4k nucleotides (nt), with hair-pin structures at both ends of each strand. The genome encodes a major nonstructural protein NS1(nt435-2448), which consists of671amino acids with a molecular mass of74kDa. Many studies demonstrated that the NS1could directly bind to the viral p6promoter and trans-activates p6promoter to regulate viral DNA replication. Other related studies reported that the NS1also trans-activated several cellular promoters to stimulate the expression of TNF-a and IL-6. In addition, the NS1caused cell cycle arrest at Gl and S phase as well as apoptosis of erythroid cells. All the data suggested that the NS1not only entered the nucleus to bind with DNA and regulated the activity of promoters, but also located in the cytoplasm to interact with potential proteins and ultimately stimulated the expression of cytokins and apoptosis-related proteins. Some results showed that the NS1mainly located in nucleus in permissive cells while quite a few in cytoplasm. However, while expressed in non-permissive cell lines, the NS1located completely in cytoplasm. Interestingly, the NS1is proved to be a protein which shuttles between nucleus and cytoplasm. However, little is known about the mechanism of this shuttle protein. The present study mainly focused on this possible shuttle mechanism of the NS1and the main research approaches and results are summarized as follows:(1) To confirm that NS1shuttles between nucleus and cytoplasm. We transfected HEK-293T with B19infectious clone pB19-4244and treated the cells with LMB after24hours of transfection to examine the localization of NS1. Here, HEK-293T transfected with the recombinant vector NS1-pEFGP-N1was used as control.(2) To find new potential NESs, we analyze the NS1protein sequence with NES-related softwares and constructed a series of NS1truncations according to the predictions. Then we cloned the different NS1truncations into pEGFP-N1vector and transfected these constructs into HEK-293T. The localization of each truncated NS1was observed with fluorescence microscope to identify the potential position of three NESs on NS1.(3) We cloned the three NESs into the pEGFP-N1vector respectively. HEK-293T cells transfected with these recombinant vectors were observed with fluorescence microscope to confirm whether all NESs are active. The point mutations were made in these NESs respectively to determine the key amino acids in each NES signal. Finally, we mutated key amino acids of NESs in the whole NS1protein sequence to see the localization differences between WT NS1and the mutants.(4) To identify which NESs are CRM1-dependent, we used LMB to treat the transfected cells and detected the direct interaction between NS1and CRM1using mammalian-two hybrid system.Our results showed that theNSl localization, in the HEK-293T cells transfected by infectious clone pB19-4244treated with LMB was changed from cytoplasm to nucleus. The similar results were observed in cells transfected with NS1-pEGFP-Nl, indicating that the NS1could shuttle between nucleus and cytoplasm and its export was CRM1-dependent. Then, based on the localization of a series of truncated and point mutant proteins, we identified two active NES, namely NES1and NES2. With the LMB treatment, we preliminarily identified that NES2but not NES1was CRM1-dependent. The analyses of the results of mammalian-two hybrid assay showed that the NS1interacted directly with CRM1, suggesting that the export of NS1was CRM1-dependent. Our results elucidated preliminarily the shuttle mechanism of NS1and gave further insights into the multiple functions of NS1protein and infection mechanism of B19virus.