Cellular entry of Hantaan virus A9 strain: specific interactions with beta3 integrins and a novel 70kDa protein.

Cellular entry of pathogenic hantaviruses had been shown to be mediated by beta3 integrins. However, no direct evidence exists that hantavirus binds to beta3 integrins, and integrin beta3 subunit is not expressed on some cells permissive to hantavirus infection. In this report, utilizing beta3-integrin-transfected CHO cells, we demonstrated that integrin beta3 subunit renders CHO cells susceptible to Chinese Hantaan virus (HTN) strain A9 (isolated in China), and the viral infection was correspondingly inhibited by antibodies to alphavbeta3, alphaIIbbeta3, beta3, and alphav integrins. Furthermore, virus overlay protein-binding assay and 'quarternary Western' analysis indicate that HTN A9 directly interacts with beta3 integrins and an unidentified 70kDa protein. These findings indicate that beta3 integrins play a crucial role in cellular entry of HTN A9 via specific interactions with the virus. In addition, a novel 70kDa protein may serves as a candidate receptor or alternative cellular component for interaction with HTN.

[The preparation and the in vitro release of OANO-1 microspheres].

OBJECTIVE: To prepare OANO-1 microspheres and test their release in vitro. METHOD: OANO-1 microspheres were made by W/O/W-liquid drying process. The surface morphology of the microspheres was observed by SEM. The mean diameter and the size distribution of microspheres, the drug loading and the incorporation efficiency were examined. The release of OANO-1 microspheres in vitro was examined by small cup method. The accumulated release percent of OANO-1 microspheres was examined. RESULT: The OANO-1 microspheres were regular in their morphology. The average particle size was 8.59 microm with over 90% of the microspheres being in the range of 1-12 microm. The drug loading and the incorporation efficiency were 48.39% and 19.32% respectively. The accumulated release percent of OANO-1 microspheres was 78.4% after 108 h. The release half-life t1/2 was 40.8 h and Higuchi equation was Y = 0.1326 X - 0.4782, r = 0.9951. CONCLUSION: The preparation of OANO-1 microspheres was well. The release in vitro of OANO-1 microspheres showed significant sustained release.

[The relationship between cellular infection by Hantaan virus and expression of beta3 integrin].

OBJECTIVE: To investigate the relationship between cellular entry of Hantaan virus (HTNV) and expression of beta3 integrin in beta3-integrin-deficient and HTNV-insusceptible China hamster ovary (CHO) cells. METHODS: Eukaryotic expression vector encoding human integrin beta3 and eukaryotic expression vector harboring human integrin alphav or alphaIIb subunit cDNA were transfected into HTNV non-permissive CHO cells individually or collectively. Screening for stable transfectant clones was performed using G418 selective (culture medium. The exogenous gene expression was analyzed qualitatively and quantitatively by immunofluorescence assay (IFA) and flow cytometry (FCM). Various modified CHO cells and untransfected CHO cells were infected using HTNV A9. At various time points after infection, HTNV antigens in infected cells were detected qualitatively and quantitatively by IFA, FCM. RESULTS: Highly-effective surface expression of beta3 integrin was measured in CHO/alphavbeta3 and CHO/alphaIIbbeta3, while weaker surface expression was detected in CHO/beta3 (P < 0.05). Expression of alphav or alphaIIb integrin in the individually transfected group was significantly lower than in the cotransfected group (P < 0.01) and the sites of localization changed. In contrast, effective surface expression was not seen when pcDNA3 was transfected alone. The infection rate of CHO/alphavbeta3 (60.1%) and CHO/alphaIIbbeta3 (55. 9%) cells were significantly higher than that of CHO/beta3 (38.7%) cells, while the infection rate of CHO/beta3 was significantly higher than that of CHO/alphav, CHO/pcDNA3 and CHO cells respectively. There was a close relationship between the positive percentage of HTNV A9-infected cells and expression of beta3 integrin. CONCLUSION: These results indicated that cellular entry of HTNV was related to the expression of beta3 integrin.

[Construction of human integrin beta3 subunit eukaryotic expression vector and efficient surface expression of integrin alphaIIbbeta3].

AIM: To establish efficient surface expression of Homo sapiens integrin alphaIIbbeta3 in beta3-integrin-deficient and HV-insusceptible CHO cells, so as to lay the foundation for the further study of cellular entry of hantavirus mediated by beta3 integrins. METHODS: Eukaryotic expression vector pcDNA3.1-beta3 harboring ORF region of human integrin beta3 subunit cDNA was constructed, then pcDNA3.1-beta3 and eukaryotic expression vector pBJ1-alphaIIb containing human integrin alphaIIb subunit cDNA were transfected into CHO cells alone or together. The expression of exogenous genes were analyzed by indirect immunofluorescence assay (IFA). RESULTS: The eukaryotic expression vector pcDNA3.1-beta3 was constructed successfully. IFA examination showed that surface expression of integrin alphaIIb beta3 was highly effective in cotransfection group, while surface expression was weak on CHO cells transfected with pcDNA3.1-beta3 alone,and no effective surface expression was found in pBJ1-alphaIIb-transfected group. CONCLUSION: Efficient surface expression of integrin alphaIIb beta3 requires expression of both subunits.