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. 2015 Feb:98:121-6.
doi: 10.1016/j.rvsc.2014.11.003. Epub 2014 Nov 18.

Domain I and II from newly emerging goose tembusu virus envelope protein functions as a dominant-negative inhibitor of virus infectivity

Dongmin Zhao  1 , Xinmei Huang  1 , Yuzhuo Liu  1 , Kaikai Han  1 , Jingfeng Zhang  1 , Jing Yang  1 , Xingxing Xie  1 , Yin Li  2
Affiliations

Domain I and II from newly emerging goose tembusu virus envelope protein functions as a dominant-negative inhibitor of virus infectivity

Dongmin Zhao et al. Res Vet Sci. 2015 Feb.

Abstract

Flavivirus envelope protein locates at the outermost surface of viral particle and mediates virus entry and fusion infection, and domains I and II of E protein play an important role in this process. In this study, we have expressed and purified goose tembusu virus (GTV) E protein domains I and II (DI/II) from E. coli, and tested conceptual approach that purified protein serves as anti-viral reagent. We found that DI/II inhibited GTV JS804 infection in BHK-21 cells in a dose-dependent manner, and this inhibition activity was achieved by binding to cell membrane specifically. Moreover, JS804 treated with DI/II specific anti-serum decreased its infectivity to BHK-21 cells. Taken together, this is first to show that the purified DI/II domain of tembusu virus expressed in E. coli was able to interfere with virus infection, which opens an avenue to develop novel anti-viral regents to prevent and eventually eradicate GTV infection.

Keywords: Domain I and II; Envelope protein; Goose tembusu virus; Inhibition.

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Figures

Fig. 1
Fig. 1
Expression of recombinant GTV E DI/II protein and detection by western blot. (A) Induction of DI/II from E. coli. Lane 1, molecular weight marker; lane 2, crude lysates from E. coli (BL21) with pET-28a vector; lane 3–5, crude lysates protein from E. coli (BL21) with pET-DI/II after IPTG induction for 0, 4, 6 h, respectively. (B) Detection of E DI/II protein after purification. Lane 1, soluble supernatant after cell sonication; lane 2, pellet fraction after cell sonication; lane 3, molecular weight marker. (C) Detection of purified GTV E DI/II with anti-E monoclonal antibody. Lane 1, molecular weight marker; lane 2, the recombinant E DI/II protein is recognized by the anti-E monoclonal antibody. (D) Detection of purified GTV E DI/II with anti-FLAG antibody. Lane 1, molecular weight marker; lane 2, the recombinant E DI/II protein is recognized by the anti-FLAG antibody.
Fig. 2
Fig. 2
Inhibition of GTV entry by DI/II protein. BHK-21 cells were first incubated with different concentrations of proteins. GTV was added and assayed for virus entry by reverse transcriptase real-time PCR assay. Data were presented from three independent experiments and statistic analysis was done with SPSS software. * Denotes statistically significant difference and ** denotes statistically extremely significance.
Fig. 3
Fig. 3
DI/II binds to the membrane of BHK-21 cells. BHK-21 cells were incubated with BSA (A), DIII (B), recombinant DI/II protein (C) and entire E protein (D) at 4 °C for 1 h and then washed repeatedly by PBS. The bound proteins were detected by anti-E monclonal antibody and visualized with fluorescence microscopy. Bars = 50 µm.
Fig. 4
Fig. 4
Plaque neutralization of GTV JS804 with murine polyclonal antibodies against DI/II protein. Antiserum was diluted in serial twofold in DMEM, each dilution (from 1:2 to 1:2048) was mixed with 200 TCID50 of JS804 virus and incubated at 37 °C for 1 h and the residual infectivity of JS804 virus was determined by plaque assay. Data were presented from three independent experiments and statistic analysis was done with SPSS software.
Fig. 5
Fig. 5
Cytotoxic assay. CCK-8 assays for cell viability were performed after 48 h incubation with 100 µg/ml of DI/II, DIII, E and BSA. The medium without cells was taken as blank control. Data were presented from three independent experiments and statistic analysis was done with SPSS software.
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