| Hantaviruses are the major causative agents of human hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), which have been becoming one of global serious issues for human public health. Hantavirus possesses a tripartite, single-strand RNA genome, with segments designated as small (S), medium (M) and large (L) encoding the viral nucleocapsid protein (NP), envelope glycoproteins (Gl and G2) and RNA polymerase L protein, respectively. The viral NP is involved in the virus replication, stimulation of the protective cell immune response and regulation of apoptosis pathway.Hantaviruses Z10 and Z37 are two important Chinese vaccine strains, which were isolated from the serum of a HFRS patient and apodemus agrarius, and serologically grouped into HTNV and SEOV type, respectively. To study the evolutionary relationship of these two Chinese vaccine strains to other recognized hantaviruses, the cDNAs encoding complete NPs of Z10 and Z37 were cloned and subsequently sequenced. Phylogenetic analysis of the NP sequences at both nucleotide and deduced amino acid levels revealed that Z37 strain is closely related to SEO virus Chinese strain R22 and AH09 and even UK strain IR461, whereas Z10 strain is genetically distinct from the HTNV Korean strain 76-118 and Chinese isolateA9, forming a sub-lineage between HTNV and SEOV serotype. This data suggest that HTNV serotype with geographic differences is more variable than SEOV.To obtain soluble and native NPs, the sequences encoding NPs of Z10 and Z37 were expressed in E.coli at 18 with 100 mM IPTG overnight and the His- tagged recombinant NPs were then purified with Ni-NAT affinity chromatography and DEAE ion exchange chromatography by using a FPLC system. The purified recombinant NPs of Z10 and Z37 were distinguished by trypsin incomplete digestion, followed by western blot using anti-His monoclonal antibody.A unique structure feature of the viral genome is a highly conserved about 18-nucleotide inverted repeat sequence is present at the terminals of each gene segment in different types of Hantaviruses, which can form a special hairpin structure when it got complemented. However, the function of the hairpin structure has not been clearly known. Electrophoresis mobility shift assay (EMSA) showed that both NPs of Z10 and Z37 hantaviruses bound specifically to the double strand DNA probe possessing the 18- nucleotide inverted repeat sequences in vitro, suggesting the inverted repeat sequence at the ends of each gene segment could be an important target site for nucleocapcid protein, and therefore plays an important role during Hantavirus packaging the viral apparatus into a complete viral particle.To investigate the intracellular NP expression and translocation, the NP coding sequence of hantavirus Z10 was introduced into NEH 3T3 cells through a retrovirus MSCV system. The integration of NP gene in the selected cell clones was confirmed by PCR and southern blot, and the expression of NP was detected by western blot. IFA in combination with laser scanning confocal fluorescent microscope shows the NP predominantly locates and partially polymerizes into inclusion bodies in the cytoplasm and membrane region of nucleus in selected cells.Macrophages are one of the target cells of Hantavirus at the early stage of infection. To explore the possible roles of NP during hantavirus infection in macrophages, the effects of NP on a mouse macrophage lineage J774 were studied. Itwas found that over-expression of Z10 NP had very little effects on J774 cells compared to the control cells, whereas there was an obvious inhibitory and cytopathic effects on macrophages J774 by stimulating with E.coli produced recombinant NPs of both Z10 and Z37 at a concentration of 2 mg/ml. These data indicate that hantavirus NP has cytopathic effects on macrophages as a extracellular stimulating signal.
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