The Up-Regulation Of CAR And DAF During CVB3 Invading Into HeLa Cells

ObjectiveCoxsachievirus, genera of family Picornaviridas and Enterovirus, exists extensively in the natural world. Coxsachievirus has 30 serological types, according to the difference of pathopoiesis characteristic to suckling mice and cellular sensitivity, Coxsachievirus is divided into two groups (A and B).The group of B has six serological types(from B1 to B6),which can provoke communicable costalgia , meningoencephalitis, myocarditis, hepatitis, hemolytic anemia and so on. After Coxsachievirus B3 (CVB3) penetrated into organism, it could produce viral myocarditis and meningitis. From the infected dynamics of CVB3, we know that CVB3 can infect many kinds of cells, which mostly owing to the interaction between Coxsachievirus and receptors of cell surface. According to analyze the difference between susceptible cells and non-susceptible cells, we found the virus receptors including coxsackievirus and adenovirus receptor (CAR), decay accelerating factor (DAF) and so on. Almost every kind of somatic cell surface expressed CAR, which may be related with cell splicing and multiplication, but the exact biological function of CAR didn't know up to now. Recently, we found that CAR was the common receptor of coxsackievirus and adenovirus, so many researchers pay attention to it. DAF extensively express at many kinds of body cells. In early discovery, DAF was a complement regulatory protein, and DAF's function of complement regulation was clearly, but the function of DAF as a virus receptor was researched very few. At present, pathogenic mechanism of CVB3 isn't clearly. During CVB3 binding with receptor, allocation of binding site, intracellular signal transduction and diversity of cystoskeleton are worth to research extremely.In our experiment, using CAR antibody and DAF antibody effect on HeLa cells, then testing the function of them to protect cells during CVB3 infection, so we can indirectly found the function of CAR and DAF during CVB3 invading into cells. At the level of RNA and protein, we test the regular pattern of CAR and DAFfollowing by virus infective time and density.Methods1,The protective function of CAR antibody and DAF antibody to HeLa cells:Cells were cultured in 5 groups:①virus group: CVB3 effected on HeLa cells as positive control.②DAF antibody group: DAF antibody interacted with HeLa cells before using CVB3.③CAR antibody group: CAR antibody interacted with HeLa cells before using CVB3.④DAF antibody and CAR antibody group: DAF antibody and CAR antibody interacted with HeLa cells before using CVB3.⑤normal group: normal HeLa cells as negative control to other groups.(1)MTT method detect cytoactive: test the optical density value of cells under the wavelength of 490nm, then calculate the mean of every group, relative survival rate of cells and inhibition ratio of antibody to virus.(2) Plaque counting method detect the influence of virulence: count the number of virus plaque, then calculate the plaque-forming unit of every group.2,RT-PCR: total RNA was extracted from HeLa with Trizol for reverse transcription and proliferation of cDNA according to the instruction. Data were analyzed by computer and the results were expressed in optical density ratio of CAR or DAF gene toβ-actin.3,Flow cytometry detection: CVB3 effect on HeLa cells first, then interacting with fluorescent antibody of CAR or DAF, in order to test the expressive condition of them.Results1,Detection of MTT method: at the survival rate of cells, the difference between D group and V group has not statistically significant; the difference between C group and V group has statistically significant (P<0.05); the difference between C+D group and V group has noticeable statistically significant (P<0.01). At the inhibition ratio of virus, C+D group is the best, which is bigger than the sum of C group and D group.2,Detection of Plaque counting method: because of the blocking function of antibody, virulence of CVB3 was descent, and the number of virus plaque decreased obviously in C+D group.3,Detection of RT-PCR and flow cytometry: after CVB3 effecting on HeLa cells, the expression of CAR and DAF were up-regulation, which compared with normal group had noticeable statistically significant (P<0.01) . After one hour, the expression of DAF came to normal, but the expression of CAR still maintain high level. The up-regulation of CAR and DAF were positive correlated with virus density.ConclusionsDuring CVB3 invading into HeLa cells, the function of CAR was bigger than DAF's as a virus receptor, and CAR and DAF maybe have the synergistic effect. After CVB3 effecting on HeLa cells, the expression of CAR and DAF were up-regulation, which were positive correlated with virus density. After CVB3 invading into HeLa cells, the expression of DAF on cell surface changed to normal, which maybe correlate with virus escaping immunologic surveillance.