The Preliminary Study On Fibronectin In The Entry And Pathogenesis Of Hantaan Virus

Hantaviruses are defined as a unique genus of segmented negativesingle-stranded RNA enveloped virus belonging to Bunyaviridae. Hantaviruscauses two kinds of infectious diseases: one is hemorrhagic fever with renalsyndrome (HFRS) which is a main diseases needed to be prevented and treatedin our country, the another is Hantavirus pulmonary syndrome (HPS) which ismainly prevalent in America.As a kind of acute viral infectious diseases, fever, hemorrhage and damageof renal function are the main symptoms of HFRS. When the body is infected byHantavirus, vascular endothelial cells and many kinds of immune cells areinvaded, which leads to increase of cytokines production, disorder of immunesystem and hyper-permeability of vascular endothelium and so on. But theunderlying mechanisms are still unknown and further researches are needed. In general, one kind of virus can only infected one type or some typesofcells as their target cells. In the initial phase of infection, specific molecularcalled viral receptor on the surface of infected cells is important, which canmediate the recognition and binding of virus and cells. β3-integrin is recognizedas the receptor of pathogenic Hantaviruses and plexin-semaphorin–integrin (PSI)domain is the site of the action. However, it is recently reported that Hantavirusinvading cells also can be mediated by clathrin. But internalization ofHantavirus can’t be inhibited completely by inhibition of clathrin whichdemonstrated that there are other ways mediating internalization of Hantavirus.Recent years in our lab, using Ras recruitment system we found that theglycoproteins G1and G2of Hantavirus interact with fibronectin (FN). It hasbeen demonstrated that FN play an important role in process of virus invadingcells.Are there other ways by which Hantavirus invade cells? What about therole of FN in the procedure of Hantavirus infecting cells? Can it promoteinternalization of Hantavirus? To answer these questions we did the researchesas follows.1. Co-localization of FN, nucleocapsid protein, caveolinRecent study reported that clathrin dependent way and caveolin dependentway were important ways by some kind of viruses invading cells. To demonstratethe role of FN in the procedure of Hantavirus invading cells, we detectedco-localization of FN, caveolin. First, we detected virus titre using double-antibodysandwich ELASA and plaque reduction nuetralization test was used as control. HUVECwas infected by Hantavirus, and the co-localization of FN, caveolin observed byLaser confocal microscopy. We found that HTNV NP, FN, and caveolinco-localized on the membrane of HUVEC. These results demonstrate that the endocytosis pathway can be enter cells by clathrin dependent way may be also bythe FN-mediated caveolin way.2. Competitive inhibition test of different segments of FN onHantavirus infectionTo clarify structural basis of FN action in the procedure of Hantavirusinvading HUVEC, five kinds segments of FN which were overall length FN,cell binding segment, connected region segment, fibronectin fragment III1-C,heparin sulfate binding segment, were used to do competitive inhibition test onHantavirus infection. Using ELISA to detect virus titer, the results demonstratedthat all these five kinds of FN segment could inhibit Hantavirus infection.Especially inhibitory function of connected region segment was dosage-dependent and other four kinds of segment were not.3. Dynamic change of FN and TGF-β1in peripheral blood of differentphases of HFRS patients.To clarify action of FN and TGF-β1in Hantavirus infection,260serum orplasma samples of HFRS patients and30normal controls in different phases werecollected. FN and TGF-β1levels were detected in different stages of HFRSpatients by ELISA and further confirmed by Western blot. The fluctuatedelevation of FN was observed in the course of the disease. FN level were higherin diuresis stage and convalescent stage than the normal controls in plasma. Andin serum FN level were lower in every stages than the normal controls whichcame to the highest in diuresis stage. The fluctuated elevation of TGF-β1levelwas observed and TGF-β1level reached the highest in hypotension/shock stagein plasma and serum.In conclusion, FN play an important role in Hantavirus internalization bycaveolin dependent way.Caveolin dependent way were important ways by some kind of viruses invading cells and the heparin binding site may play a key role.Atthe same time, the virus infected with the organism can cause FN changes, andTGF-β1and FN requires further investigation.These results provid basis forfurther researches on molecular mechanism of Hantavirus diseases.