| Hantaviruses are enveloped viruses which define a unique genus within the Bunyaviridae and contain a tripartite negative-stranded RNA genome. The viral RNA segments, L, M and S, encode an RNA dependent RNA polymerase (RDRP), two envelope glycoprotein (GP, G1 and G2), and nucleoprotein (NP), respectively. Gl and G2 are thought as the virion attachment protein (VAP) exposed on the virions surface. Pathogenic hantaviruses mainly cause two kinds of acute diseases: one is hemorrhagic fever with renal syndrome (HFRS), which is charactered by fever, vascular hemorrhage, kidney dysfunction and immunologic dysfunction, another is hantavirus pulmonary syndrome (HPS), which is charactered by pulmonary infiltration, pulmonary interstitial edema and quickly developed respiratory distress and failure. In China about 50,000 -100,000 people per year were infected by Hantavirus with the severe symptoms and high mortality. There are no therapeutic approaches for hantaviruses diseases, and the etiologic mechanism of hantaviruses infections have not yet been defined.The first step in a viral infection and propagation is binding of the virus to cell surface molecules. It is one of the main factors that determining virushost specificity, tissue-tropism and pathogenicity. Therefore, the studies about Hantavirus receptor may help to explain the etiologic mechanism and to find useful new strategy for antivirus treatments and new potent vaccines.At present, it has been reported that integrin β3 maybe one of the receptors of HFRS/HPS-associated hantaviruses. However, the receptor of Hantavirus is still not completely defined. First, HTNV is causes of HFRS and SNV is causes of HPS, they however share the same receptor integrin β3. Second, the antibody and ligands of integrin β3 can not inhibit the infectivity totally. So there should be some others receptors or coreceptors exist on the cells. At the same time, the specificity of integrin β3 subunit as Hantavirus receptor needs to be further investigated.In this study, we explored the new type yeast two-hybrid system—RRS system, and use G1 and G2 as baits to screen preys out from cDNA yeast expression library. Meanwhile, further studies to elucidate the interactions of integrin β3 and G1/ G2 are needed.1. In order to construct human umbilical vein endothelial cell (HUVEC) cDNA library, total RNA and purified mRNA were extracted from human umbilical vein endothelial cell line HUVEC. cDNAs were synthesized though the cDNA synthesis kit and cDNA fragments smaller than 500bp and redundant EcoR I adaptors were removed. Then, cDNAs were connected with the yeast expression vector pYesMApolyA. The results showed that the HUVEC cell line cDNA library consisting of 5.2x 105 recombinants had been constructed successfully. The average exogenous inserts of the recombinants were larger than 1.6kb. The constructed cDNA library is suitable to screen target genes.2. Met-G1, Met-G1, Met-G2 和 Met-G2'were constructed successfully and the results of activations test showed that all of the four baits could be used to screen target genes..3. The cDNA library was hybridized with two kinds of baits vectors Met-G1 and Met-G2, respectively. The ligands were sequenced and then analyzed on BLASTP. The results showed that one recombinant of Gl ligand and two recombinants of G2 ligand were obtained. The Gl ligand was similar to containing EH1 domain protein. The EH domain has been shown to be an important motif in proteins involved in protein-protein interactions, endocytosis, vesicle transport and signal transduction. It implicated that this recombinant associated with Hantavirus endocytosis. The G2 ligands were similar to TNFRSF1A and RNP24. TNFRSF1A was Tumor necrosis factor receptor superfamily member 1A precursor contained TNFR domain and Death domain. TNFRs triggered multiple signal transduction pathways. They were involved in inflammation response, apoptosis, and autoimmunity. RNP24 belonged to p24 family, and members of this family were implicated in bringing cargo forward from the ER and binding to coat proteins by their cytoplasmic domains. So this two ligands might be associate the infection and maturation of Hantavirus.4. The recombinants were constructed successfully, including pVpDisplay, β3-pYesMApolyA, β3EGFP-N3, G1-pDisplay, G1-EGFP-N3, G2-pDisplay and G2-EGFP-N3. The results of reconstruction of Hantavirus-associated receptor system suggested that the TCID50 of CHO tranfected with integrin β3 compared to CHO went up 10 times higher. The results of two hybridize assay and coimmunoprecipitation assay suggested that the interactions existed between integrin β3 and G2 but did not exist between integrin β3 and G1. All results above suggested that integrin β3 was one of Hantavirus receptors.Above all, cDNA library was constructed and the library contained 5.2x105 recombinants. There existed some more complex mechanisms in attachment and internalization of Hantavirus. It suggested that the Hantavirus... |
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