| Dengue virus (DV) belongs to the family Flaviviridae and there are four closely related, but antigenically distinct, virus serotypes (DV1-4). DV infection has reemerged as a more severe illness in humans than it was in the past. DV causes classical dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DF is a nonspecific viral febrile illness while DHF/DSS is severe and fatal hemorrhagic disease. The pathogenesis of DV infection is not clear. The most characteristic feature of DHF/DSS and the best indicator of disease severity is plasma leakage, which occurs systemically, progressing quickly, but will resolve within 1-2 days in patients who receive appropriate fluid resuscitation. No discernible sequelae are usually found. It is generally thought that plasma leakage is due to altered vascular permeability and/or slight structural destruction of endothelial cells.Integrins are heterodimeric receptors composed of a combination ofαandβsubmits, and play important roles in cell-cell adhesion, cell migration and extracellular matrix protein recognition. Integrin directed cellular migration is an essential function of vascular endothelial cells (VEC) in wound repair, angiogenesis and the maintenance of vascular integrity.β1 andβ3 integrins are abundant surface receptors of VEC and platelets, and play central roles in maintaining capillary integrity. Pathogenic hantaviruses, a pathogen causing mucocutaneous hemorrhage and vascular permeability effects similar to DHF, selectively inhibitβ3 integrin directed endothelial cell migration. In addition,ανβ3 integrin is commonly used as receptors or coreceptors for many different viruses. Recently, it was reported that West Nile virus, which belongs to Flaviviridae family as DV, could also useβ3 integrin as receptor for entry into Vero cells.DV infection of human VEC was proposed in 1978. Structural alterations of microvascular endothelial cells were found in skin biopsies and viral antigens were detected in microvascular endothelial cells from the brain of a fatal case of DHF. According to the reports, we presumed that integrins might be involved in DV infection, and thereby contribute to increased vascular permeability observed in DHF/DSS. Therefore, DV-integrins interaction was investigated using human dermal microvascular endothelial cells line-1 (HMEC-1) in this study. The main results were as follows:(1) The expression ofβ3 integrin was up-regulated after DV2 infectionWe comparedβ1 andβ3 integrin receptors'level on the surface of DV2-infected and mock-infected HMEC-1 using flow cytometric analysis. The mean fluorescence intensity ofβ3 integrin, which represented the receptor level on surface of HMEC-1, showed gradually increased tendency after infection: there was no obvious change at 24hr p.i., and it increased markedly at 48hr p.i. and peaked at 72hr p.i. The highest expression level ofβ3 integrin increased by 2.54-fold compared with that seen in mock-infected cells (P<0.01, n=3). However theβ1 integrin receptor numbers showed little change on infected cells. Our previous study showed the highest viral titer with 105 PFU/ml in HMEC-1 was also observed at 72hr p.i., when cells were infected at 1 of MOI. These suggested that DV2 infection could induce increase in expression level ofβ3 integrin.Next, we used real time RT-PCR to confirm the increased expression level ofβ3 integrin. We found gradually risen expression level ofβ3 integrin mRNA after infection and it reached to peak value at 72hr p.i. with a increase by 14.48-fold (p<0.01, n=3). The change tendency of expression level ofβ3 integrin mRNA was corresponding with the results of flow cytometric analysis, further indicating that there were increase in expression level ofβ3 integrin after DV2 infection.(2)β3 integrin showed high co-localization with DV2 E proteinDV2 infected HMEC-1 were subjected to immunostaining usingβ3-JM2E5 and DV2 E protein polyclonal antibody. Mock-infected HMEC-1 showed very faint staining. After infection, there was little change in the staining intensity in HMEC-1 at 24hr p.i. With infection progress, the high intensity of staining was observed in the perinuclear regions and membrane area at 48hr and 72hr p.i., and the staining intensity showed high co-localization with DV2 E protein. In contrast, there was little change in the staining intensity ofβ1 integrin antigen in mock-infected cells and DV2 infected cells. This result might indicate close association ofβ3 integrin and DV2. (3) Blockage ofανβ3 integrin function partially inhibits DV2 infectionTo investigate effects of integrins on the viral entry, their functional blocking MAbs and physiological integrin ligands were used for DV2 entry blocking assay. Cells were pre-treated with functional blocking MAbs againstβ1,αν,ανβ3 integrin and fibronectin, vitronectin, respectively.αν-M9 andανβ3-LM609 exhibited inhibition of 30% and 38% of DV2 penetration into HMEC-1 respectively, and the binding of fibronectin and vitronectin to the cell surface resulted in inhibition of DV2 entry by 32% and 23% at 100μg/ml, respectively. There was little change of DV2 penetration whenβ1-JB1A, BSA, or mouse IgG1 was used, respectively. In the results,ανβ3-LM609 showed inhibitory effects on viral entry with higher efficiency thanβ1-JB1A, suggestingβ3 integrin might play a central role in the viral entry.(4) Solubleανβ3 Integrin Blocks DV2 Entry into HMEC-1 Cells.To further confirm the specificity ofανβ3 integrin in mediating the entry of DV2 into cells, we incubated solubleανβ3 andανβ5 integrin respectively with DV2 before overlaying this complex onto a HMEC-1 cell monolayer. Solubleβ3 integrin could effectively block DV2 infection in a dose-dependent manner. When 10μg/ml ofβ3 integrin was used, almost 100% virus entry was inhibited, while solubleανβ5 integrin at same concentration only showed 23% inhibition of virus entry. As control, BSA showed no effects on the viral entry. These results indicated the specific interaction of DV2 withανβ3 integrin, hence preventing the subsequent infection of HMEC-1 cells.(5) Gene Silencing of Humanβ3 Integrin Inhibits DV2 infectionSmall interfering RNA (siRNA) against specific sequence encoding for humanβ3 integrin was constitutively expressed from mammalian expression vector, pSilencer 3.0-H1 neo. Transfected cells were screened with G418 and single cell clones (3 for siRNA at position 1262 and 2 for siRNA at position 1932) were obtained. The down-regulation of theανβ3 integrin in each of the single cell clones was detected by flow cytometric analysis. As compared to mock-transfected cells with pSilencer vector (negative control), all of siRNA clones showed different down-regulation level ofβ3 integrin. The most striking down-regulation was seen in clone-2 of siRNA at position 1262, and the relative number ofβ3 integrin was only 38% of that in control cells. So we named this cell clone as ECV-β3 (-), and used it as host cells in DV2 entry assay. The quantity of virus entry into ECV-β3 (-) exhibited significant reduction by an extent of 89% when compared to mock-transfected cells. These results also indicated thatανβ3 integrin might be necessary for DV2 entry into HMEC-1.(6) Recombinantβ3 integrin promotes DV2 infection of CHO-K1 cells.CHO-K1 cells, which are about 100-fold less susceptible to DV2 infection than ECV304 cells or Vero cells, and the stable transfected CHO-K1 variant cell lines C9,C10,F8,H6, which express theανβ3 orαⅡbβ3 integrins, were used to determine whetherβ3 integrin expression facilitates DV2 infectivity. All the variant cell lines were more susceptible to DV2 infection than parental CHO-K1 cells.In summary, this study demonstrated that DV2 infection could induce up-regulation ofβ3 integrin, which might play an important role in DV2 entry. These observations might lead novel insights into the involvement ofβ3 integrin in pathogenesis of DV infection. However, the binding sites ofβ3 integrin with viral proteins and the transcriptional mechanisms mediatingβ3 integrin expression during DV infection remain to be defined. |
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