Cloning, Sequence Analysis And Expression Of Structure Protein Gene Of Seoul Hantavirus

Hantaviruses, the etiological agents of hemorrhagic fever with renal syndrome (HFRS) and the newly recognized hantavirus pulmonary syndromes (HPS), are classified in the Hantavirus genus in the family Bunyaviridae. The number of characterized hantavirus continues to increase and over 30 antigenically and genetically distinct viruses are currently recognised; of these only Hantaan (HTN), Seoul (SEO), and Puumala (PUU) viruses were reported in China, and they were agents of severe, common, and mild HFRS separately.HFRS, which is characterized by pyrexia, haemorrhage, and renal lesion, consists of Korean hemorrhagic fever (KHF), epidemic hemorrhagic fever (EHF), and nephropathia epidemica (NE). HFRS poses a significant public health problem in Asia and Europe with an annual incidence of 100 000 hospitalized cases. Its early reports could be traced back to the year 1913 in Soviet Union, 1932 in Northeast China, and 1934 in Sweden. Ever since Lee et al. isolated hantavirus strain 76-118 from lung tissue of an apodemus agrarius captured in epidemic area in 1978, various types of hantaviruses have been isolated successively.The outbreak of HPS in the Four Corners Region of the United States, starting with a cluster of deaths in 1993 and with a very high fatality rate in the initial outbreak, changed the recognized spectrum of hantavirus diseases. The presentation was primarily a febrile illness complicated by the rapid development of respiratory failure; renal and hemorrhagic manifestations were not pronounced. The emergence of HPS further increased world attention. Virus isolations showed HPS was caused by several hantavirus types, such as Sin Nombre (SN), New York (NY), Black Creek Canal (BCC), Bayou (BAY), and Andes (AN). No HPS case was reported in China till now.In nature, hantaviruses are maintained in persistently infected rodents and are transmitted to humans via inhalation of contaminated excreta and secreta. Each genotype has its own specific main rodent host. In Asia,Apodemus agrarius (striped field mouse) is the primary reservoir of HTN viruses, and Rattus norvegicus (brown rat) is the primary reservoir of SEO viruses.In China, HFRS caused by HTN and SEO viruses has a broad scope of prevalence with high fatality rate, thus it is a significant public health problem in China. Ninety percent of the total cases worldwide happened in China. According to the publication of the Chinese Health Ministry in 2004, 25041 cases were reported, of which 254 cases were fatal. The incidence rate increased by 10.54% compared with that in 2003. In recent years, the number of Seoul-type cases has increased slightly. Shandong Province is a very severe epidemic area of HFRS with about one third of total cases in China. Its annual incidence rate is consistently among the top three in China. The majority of the cases are caused by virus infection with Seoul-type, followed by Hantaan-type. The improvements of diagnostic and preventive methods of HFRS depend on the etiological research of HV, in which the essential and basic research is the genetic and molecular epidemiology work on HV genome. In Shandong Province, the research work on this aspect was seldom and the genome segment of only a few isolates was wholly sequenced. The further research is needed.HV is spherical enveloped particle with spikes formed by G1 and G2 glycoprotein. Inside the coat is the nucleocapsid formed by genomic RNA, nucleocapsid protein and RNA polymerase. The nucleocapsid is abundant in virus particle. HV has a tripartite, single-stranded negative-sense RNA genome. The small, medium, and large genomic RNA segments encode the nucleocapsid (N) protein, the glycoprotein precursor (GPC) with envelope glycoprotein G1 and G2 as its cleavage fragments, and the virion-associated RNA polymerase (L protein), respectively.G1 and G2 proteins play an important role in HV pathogenicity, as the viral domains for neutralization, receptor-binding, membrane fusion, and neutralization are all located in G1 and G2 proteins. The coding order of M segment is NH₂-G1-G2-COOH. The GPC is cotranslationally cleaved to generate two proteins G1 and G2, by cellular signal peptidase at the pentapeptide motif WAASA. G1 and G2 form a heterodimer anchored in the endomembrane with some transported to the plasma membrane. It is accepted that the heterodimerization of G1 and G2 is essential for their correct folding and transport to the Golgi complex, where they are both modified by N-linked glycosylation. The co-expression of G1 and G2 is essential for their transportation and function. G1 and G2 are abundant with cysteine residues, which is important in the maintenance of structure of heterodimer in endomembrane. G1 and G2 have 5-7 N-linked glycosylation sites, and both have typical signal peptide at the N terminal. The C terminals of G1 and G2 have not yet been determined, and the computer prediction showed they contain 115 and 8 amino acids, respectively. It is accepted that the C terminal of G1 is easy to be proteolysed, as is the reason for the low expression level of glycoproteins in cell. In the Golgi complex, the viron is formed and buds into the endomembrane, and is eventually released outside the cell. The mutation of M segment is the fastest in the 3 segments.N protein is the most abundant viral component in both virions and infected cells, and inclusion is easy to be formed in cells. NP is a multifunctional viral macromolecule. It encapsidates viral RNA (vRNA), interacts with the virus polymerase and glycoproteins, and interferes with important regulatory pathways in infected cells. Amino acid sequence of N protein is relatively conserved with high immunogenicity. The N-terminal domain folds into a coiled-coil structure with high hydrophilicity. Almost all types of human anti-serum can recognized this domain. In the central is located the RNA-binding domain, and it is highly conserved part that spans aa residues 175-217. In HFRS patients' sera, the anti-N antibody emerges the most quickly, and almost all patients' sera are positive in anti-N antibody after infected by HV for one week. Thus to develop the new serological diagnostic method based on recombinant N protein is one of the hot spots in the research work on diagnostic reagent development.The virus-cell or cell-cell fusion caused by HV is low-pH-dependent. Enveloped HV virions fuse their envelopes with the endosomal membrane under acidic condition and cause fusion of infected cells, which is mediated by fusion peptide exposed after the structure alteration of GPs under low pH condition. Previous study confirmed that Vero E6 cells infected with various types of HVs undergo cell-cell fusion under acidic condition. A study showed HTNV GPs acted as fusogens at the cell surface. No fusion activity was observed either in infected Vero E6 cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. No fusion promotion or attenuation was observed with co-expression of recombinant N protein. But till now, there is no research work on whether SEOV GPs act as fusogens without the interaction with N protein. Cell fusion activity is one of essential steps of virus infecion in duplication, diffusion, and pathopoiesis. Thus the study on SEOV-mediated cell fusion is of great significance.In our study, some biological methods, such as RT-PCR, gene recombination, gene cloning, sequence analysis, eukaryotic expression, SDS-PAGE, Western-blot, indirect immunofluorescence assay (IFA), were applied to: (I) clone and sequence ZB8 complete M segment and JUN5-14 S segment that encodes full-length N protein gene to study the genetic character of Shandong HV; (II) perform homologous comparison and phylogenetic analysis on M and S sequences, to illuminate the molecular epidemiological character of Shandong HV, and to investigate the mutation regularity of HV; (III) express the N protein gene in BHK-21 cells and identify the expression products, and to examine the immuno-activity of rNP as antigen substrate in IFA in detecting HFRS patient sera; (IV) construct a yeast expression vector of N protein gene and express the N protein, and to identify the rNP, in order to lay foundation for serological diagnostic reagent development; (V) construct the eukaryotic expression vector of GPs gene and express them in Vero E6 cells, to observe the cell-cell fusion under acidic conditions.1. Cloning and sequence analysis of M and S segment of Shandong hantavirusesIn order to avoid genomic alteration induced by cell culture adaptation, total RNA was directly extracted from lung tissue of rodents. The HV strains infecting the two rodents were designated as ZB8 and JUN5-14, respectively. Reverse transcription was performed, and ZB8 M segment and JUN5-14 S segment were PCR-amplified using SEO-specific primers. PCR products were ligated into T vector, and the ligation products were transformed into competent E. coli DH5α. DNA sequencing confirmed the correct insertion of HV genomic segments. Cloning of M and S segment lay foundation for development of new diagnostic reagent and genetic engineering subunit vaccine.Analysis on the sequence character of M and S segments was performed. Full-length M segment of ZB8 consisted of 3 651 nucleotides in length with 1107 adenines, 1 097 thymines, 682 cytosines, and 765 guanines, and encoded a protein of 1 133 amino acids. Full-length NP gene of JUN5-14 consisted of 1 290 nucleotides in length with 403 adenines, 292 thymines, 257 cytosines, and 338 guanines, and encoded a protein of 429 amino acids. The information on the transmembrane location and secondary structure of GP and NP was predicted via TmPred and DNAStar software.Homologous comparison was performed with other HV strains. ZB8 had 84.1-99.0% identity with other SEO isolates at nucleotide level of M segment. And JUN5-14 had 87.6-97.8% identity with other SEO isolates at nucleotide level of S segment. In the complete M segment nucleotide sequence, mutations in 3'NCR were most frequent, while sequence in 5' NCR was most conserved. Although Shandong HV and other SEO viruses were relatively distinct at nucleotide level in the M and S segments, they were highly conservative at the protein level with 96.9-99.6% and 98.1-99.8% homology in the M and S segment, respectively.Phylogenetic analysis was performed by applying the Neighbor-Joining method using Mega3 software. ZB8 and JUN5-14 were both grouped into Subtype 3 of Seoul hantavirus, the members of which included Z37, ZT10, ZT71, BjHD01, 93HBX12, and Zy27. In the M tree, ZB8 had the most intimate relationship with GM04-38, which was also Shandong strain. Sequence analysis investigated the mutation regularity of Shandong HV, and provided molecular foundation for diagnosis and prevention of HV.2. Expression of NP gene of JUN5-14 in BHK-21 cells and antigenicity analysis of recombinant NPThe NP gene was PCR amplified and subcloned into the expression vector pBluscript II SK⁺ with Pst I and Sac I restriction sites under the control of T7 promoter, and the resultant expression construct was designated as pBSK-NP. NP gene was expressed in BHK21 cells using the T7 RNA polymerase expression system. After incubation at 37℃for 48h, the cells were washed three times with phosphate buffered saline (PBS) and resuspended in PBS for IFA and Western blotting analysis. Indirect IFA using positive serum derived from a patient with HFRS showed that NP gene was adequately expressed in the transiently transfected BHK21 cells, and the fluorescent signal was robust and concentrated in the perinuclear region of the transfected cells. SDS-PAGE and Western blotting detected a specific band with a molecular weight of 48 kDa that is consistent with the predicted molecular weight of the NP.In IFA, expressed NP of JUN5-14 in BHK21 cells served as antigen in detecting IgG in patient sera. Sixteen out of the seventeen samples were positive for antibodies against NP, while no negative control sera samples show positive results. The data indicated that the expressed rNP of JUN5-14 could serve as an adequate antigen for sensitive detection of anti-NP IgG in patients' sera, which lay foundation for stable expression of NP gene and development of diagnostic reagent.3. Expression of hantavirus JUN5-14 strain NP gene in the yeastThe whole sequence of NP gene was amplified by PCR, and corresponding restriction enzymes sites were created in both ends of the sequence. PCR products and plasmid pGAPZαA were double digested, ligated and transformed into E. coli DH5α. The products were screened by Zeocin, and after being confirmed by enzyme digestion and sequence analysis, the pGAPZ-NP was conducted.Plasmid pGAPZ-NP was digested with Avr II and transformed into Pichia pastoris GS115 cells with the help of LiCl. The products were screened for twice on the YPD plate with 100μg/ml of Zeocin. Single clones were selected and propagated in a large amount. The expression products collected in different periods of culture were characterized with SDS-PAGE and Western blot.SDS-PAGE showed that the recombinant N protein was expressed efficiently and constantly in Pichia pastoris GS115 cells. The quantity come to a peak at 48h post culture, and stabilized hereafter. Protein expression existed in both supernatant and cells. Western blot showed that the protein in both supernatant and cells could react with the anti-HV positive serum, as confirmed the NP gene was expressed successfully in Pichia pastoris GS115 cells and had a good immunological characteristic.4. Transient expression of glycoproteins G1 and G2 in Vero E6 cellsHantavirus ZB8 G1 and G2 gene were cloned into plasmid pCAGGS/MCS, respectively. After being confirmed by enzyme digestion and sequence analysis, the pCAGGS/G1 and pCAGGS/G2 were conducted. Then the two vectors were transfected into Vero E6 cells. After treated with acidic MEM, the cells were fixed and stained by Giemsa and were observed under microscope. An IFA was also performed to detect the expression of glycoproteins.IFA showed the G1 and G2 gene was successfully expressed, and the fluorescent signal was robust and concentrated in the perinuclear region of the transfected cells. Giemsa staining showed cell-cell fusion could be induced under the co-expression of G1 and G2 at acidic conditions. The co-expression of NP gene could not enhance the fusion. Our study confirmed first that the glycoproteins G1 and G2 were the fusogen that induced hantavirus cell fusion at the genetic level, and lay foundation for the study on the structure and function of glycoproteins.The results suggested as followings:The results above prove that the hantavirus ZB8 complete M segment and JUN5-14 S segment were cloned successfully. They are both subtype 3 of SEO virus. NP gene was expressed successfully in BHK-21 cells and the rNP possessed good immunoreactivity. And the high sensibility and specificity of rNP in detecting anti-HV antibodies in HFRS sera via IFA confirmed that JUN5-14 NP is a good target antigen for the diagnosis of HV infection. NP gene expressed very well in the yeast expression system, and the rNP possessed good immunoreactivity, so it may be a candidate for the coating antigen of ELISA. G1 and G2 genes were expressed successfully in Vero E6 cells. Co-expression of glycoproteins could induce cell-cell fusion under acidic conditions. Our work investigated the mutation regularity of Shandong HV, and provided good foundation for further study on the development of diagnostic reagent and subunit vaccine.