Preliminary Study On The Molecular Echanism Of Cell Fusion Induced By Hantavirus

Background And Obiective of ResearchHantavirus is a member of the Bunyaviridae family, and has a tripartite, single-stranded negative sense RNA genome. The small (S), medium (M), and large (L) genomic RNA segments encode the nucleocapsid protein (NP), the precursor to the virion envelope glycoproteins (GPC), and the RNA-dependent RNA polymerase, respectively. GPC is cotranslationally cleaved to generate two proteins designate G1 and G2 which are modified by five N-linked glycosylation, four sites on G1 (N134, -235, -347, and -399) and the only site (N928) on G2. In general, N-linked oligosaccharide chains can affect protein functions in many ways, including promotion of proper folding, maintenance of protein conformation and stability, protection of proteins from proteolysis, and modulation of the biological activities of proteins. China is the most seriously affected country by Hantavirus in the world, almost 90 percent hemorrhagic fever with renal syndrome (HFRS) cases of the world occur in China. In our country, HFRS mainly causes by two etiological agents Hantaan virus (HTNV) and Seoul virus (SEOV), so we selected these two type viruses as research targets.In general, fusion of the viral envelope with the target cell membrane is an essential step in the entry of enveloped viruses into cells. This membrane fusion process leads to the release of viral proteins and the RNA genome into the host cell, initiating an infectious cycle. Enveloped viruses not only can mediate fusion between the viral envelope and host cell membrane, but also can mediate cell-cell fusion. Studies on other enveloped viruses indicates that glycoproteins (GP)s (I.e. fusion protein) on the surface of viruses are responsible for these membrane fusion processes. Fusion proteins contain a short region called the "fusion peptide" that is necessary for membrane fusion. In general, the process of membrane fusion involves dissociation of native protein complexes, exposure of fusion peptide and insertion into the pericellular membrane. The energy released during this transition from metastable intermediat at the viral surface to stable target membrane-inserted complex is used to drive the merging of the viral envelope and cell membranes.Membrane fusion mediated by fusion proteins is the most important step in the entry of viruses into host cells, and elucidation of this process would favor the emergence of drugs and vaccines to block viral infection. Dissection of the viral membrane fusion process has led to a new strategy in others enveloped viruses therapy development-targeting viral entry. For example development of enfuvirtide for prophylaxis against HIV infection is based on the membrane fusion process mediated by gp41 of HIV. Furthermore, because many enveloped viruses likely use the same mechanism of entry, similar therapeutic strategies may be effective against a wide range of viral diseases.As an enveloped virus, when HTNV infects a host cell, its membrane must fuse with the host cell membrane, allowing the contents of the virus to be transferred to the host. Although the cell fusion activities mediated by Hantavirus under acidic conditions were discovered over 20 years ago, little is known about the exact location of fusion peptide or the mechanism involved. Recently, researchers reported that envelope glycoproteins of HTNV maybe participate the process of fusion and considered that GPs is the fusogens of HTNV. Moreover, proteomics computational analyses suggest that Bunyavirus G2 proteins are viral fusion proteins, with a structure similar to the fusion proteins of Alphaviruses and Flaviviruses. Furthermore, there is an internal domain which is conserved and in similar locations in G2 of viruses in each genus of the Bunyaviridae. These results suggest that the internal domain is likely to be the fusion peptide of viruses. But these hypotheses are still deficient of evidence to surport. In this study, we approached the mechanism of the process at a molecular level and provided the direct evidences to support these hypotheses. Part I: Preliminary Identification of Gene Segment on Fusion of Hantavirus Structual ProteinsIn the first part of our research we expressed NP and GPs separately or together of three different Hantavirus strains (two strains of Hantaan and one Seoul virus) in Vero E6 cells and then carried out the cell fusion experiments. The results indicated that envelope GPs of Hantavirus could mediate cell-cell fusion independently.Materials and Methods:1. The GPs and NP-encoding sequences of three different Hantavirus strains (two strains of Hantaan and one Seoul virus) were subcloned into the expression plasmid pcDNA3.0 under control of a CMV promoter.2. The recombinant GPs and NP of three different Hantavirus strains were transfected into the cells, alone or together, using Lipofectamine2000.3. After transfection Indirect Immunofluorescence Assay (IFA) and immunoprecipitation were performed to examine the viral gene expressions.4. Surface localization of viral structural proteins examined by flow cytometry.5. After expression of viral proteions cell fusion experiments and its inhibition assay were performed.Results:1. GPs and NP of all three strain viruses were expressed in Vero E6 cells.2. The cell surface expressions of GPs, but not of NP, were observed by flow cytometry.3. Syncytia formed in the cells expressing GPs regardless of NP expression, and no discernible increase in fusion was observed with the co-expression of GPs and NP.4. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not.5. Although efficiency of cell fusion differed all of three stain viruses showed the approximate results.Conclusions: 1. GPs of HTNV can mediate cell cell fusion and this process independent of other viral components (e.g., the NP) and replication.2. All of three stain viruses showed the approximate results. So we can conclude that envelope GPs as fusogens may be a common characteristic of genus Hantavirus.Part II: Role of N-Linked Glycosylation on GPs of HTNV in cell fusionIn the second part of our research we investigated the role of N-linked glycosylation on GPs of HTNV strain A9 in cell fusion. We found that the oligosaccharide chain on G2 is crucial for cell fusion. Considering that the N-linked glycosylation of HTNV GPs plays an important role in protein folding and intracellular trafficking, we believe that G2 is likely to be the fusion protein of HTNV.Materials and Methods:1. Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants. One (for a single mutation) or two (for a double mutations) of the asparagine (N) residues at the N-linked glycosylation sites on G1 and G2 were replaced with alanine (A) in order to construct the N-glycosylation site mutants. The five single N-glycosylation site mutants were designated N134A, N235A, N347A, N399A, and N928A according to the position of the substitution. Three double glycosylation site mutants were designated N235/928A, N347/928A, and N399/928A.2. The GP gene mutants were also transfected into Vero E6 cells and then immunoprecipitation and flow cytometry analysis were performed to examine the viral gene expressions and surface localizations.3. After expressions of mutants cell fusion experiments were performed.Results:1. The results of immunoprecipitation indicated that the site-directed mutation of N-linked glycosylation resulted in a significant electrophoretic mobility shift of G1 and G2, which corresponded to the apparent molecular weight losses of approximately of 3000 Da for the single-site mutants and 6000 Da for the double-site mutants. These differences are consistent with the loss of one or two N-linked oligosaccharide chains and indicated that the N-linked glycosylation were demolished.2. All of the mutated GPs were expressed on the cell surface, except for N134A, which only expressed G2.3. Considering deficiency of viral protein expression at the cell surface, no syncytium was observed with mutant N134A.4. In other mutants, mutation of glycosylation site on G1 had no effect on cell fusion, while a mutation of the single site on G2 (N928) resulted in a loss of cell fusion.Conclusion:N-linked glycosylation on G2 is essential for fusion, while lack of N-linked glycosylation on G1 had no discernible effects on cell fusion. Considering that the N-linked glycosylation plays an important role in protein folding, it is likely that G2 is the fusion protein of HTNV.Part III: Preliminary Identification of Fusion Peptide On GPs of HTNVIn the third part of our research we investigated the role of a highly conserved domain (aa763-785) on GPs of HTNV strain A9 in cell fusion by site-directed mutagenesis. We found that six mutants on the conserved domain blocked cell fusion completely. These results are the most compelling evidence for the direct involvement of the conserved domain in fusion and suggested that it is likely the fusion peptide of HTNV.Materials and Methods:1. Mutagenesis of a HTNV GPs cDNA was used to evaluate the role of a conserved domain (aa763-785) in membrane fusion. To construct the mutants, one of the amino-acid residues in the conserved domain was replaced with another residue. The eleven mutants were designated G772D, G774D, G776D, P767D, P771D, C765S, C770S, C777S, C780S, D769A and D785A respectively according to the position of the substitution. 2. The GP gene mutants were transfected into Vero E6 cells and then immunoprecipitation and flow cytometry analysis were performed to examine the viral gene expressions and surface localizations.3. After expressions of mutants cell fusion experiments were performed.Results:1. Substitution of Ala for Asp-769 or Ala for Asp-785 had no effect on fusion activity.2. Substitution of Asp for Pro-767, Asp for Pro-771 or Ser for Cys-780 abolished cell surface expression of the GPs.3. Substitution of Asp for Gly-772, Asp for Gly-774, Asp for Gly-776, Ser for Cys-765, Ser for Cys-770 or Ser for Cys-777 completely blocked cell-cell fusion activity without affecting GPs surface expression.Conclusion:The complete cell-cell fusion block in the six mutants thus is the most compelling evidence for the direct involvement of the conserved domain (aa763-785) in fusion. Here we present evidence that this domain is likely to be the fusion peptide of HTNV.