Glycogen synthase kinase 3 promotes the proliferation of porcine epidemic diarrhoea virus by phosphorylating the nucleocapsid protein

Porcine epidemic diarrhoea virus (PEDV) is a highly pathogenic porcine enteric coronavirus that causes severe watery diarrhoea and mortality in piglets. The nucleocapsid protein (N) is the most abundant viral protein and is highly phosphorylated, with the phosphorylation level directly affecting infection and proliferation. Here, we characterized the phosphorylation level of the N protein and found that its SR (Ser and Arg) motif was highly phosphorylated. The phosphorylation level significantly decreased after mutation of threonine (Thr) to serine (Ser). Through screening, it was determined that GSK3α/β plays a major role in phosphorylating the SR motif. Using GSK3α/β inhibitors or directly knocking out the GSK3α/β gene significantly inhibit PEDV proliferation. Finally, we used yeast recombination technology to develop a reverse genetics system for assessing PEDV and confirmed that no differences existed between the wild-type strain and the rescued virus. Using this platform, we generated a PEDV N protein SR motif mutant strain. We found that, compared to the wild-type strain, the proliferation of the mutant strain was significantly weakened, confirming that the phosphorylation of the SR motif is crucial for PEDV proliferation. In summary, we verified the phosphorylation sites of the PEDV N protein and the associated protein kinases, providing new insights into the development of relevant therapeutic strategies.

猪流行性腹泻病毒（Porcine epidemic diarrhoea virus, PEDV）是一种高致病性猪肠道冠状病毒，可引发仔猪严重水样腹泻并导致较高死亡率。核衣壳蛋白（nucleocapsid protein, N）是病毒含量最丰富的结构蛋白，且具有高度磷酸化修饰，其磷酸化水平可直接调控病毒的感染与增殖过程。本研究首先对核衣壳蛋白的磷酸化水平进行了表征，发现其SR基序（丝氨酸-精氨酸富集基序，SR motif）存在高度磷酸化修饰；将苏氨酸（threonine, Thr）突变为丝氨酸（serine, Ser）后，该基序的磷酸化水平显著降低。通过筛选实验，本研究确定糖原合成激酶3α/β（glycogen synthase kinase 3α/β, GSK3α/β）是介导SR基序磷酸化的关键激酶；使用糖原合成激酶3α/β抑制剂或直接敲除GSK3α/β基因，均可显著抑制猪流行性腹泻病毒的增殖。最后，本研究借助酵母重组技术（yeast recombination technology）构建了用于猪流行性腹泻病毒研究的反向遗传操作系统（reverse genetics system），并验证了野生毒株与拯救病毒之间无显著差异。依托该操作系统平台，本研究制备了猪流行性腹泻病毒核衣壳蛋白SR基序突变毒株；实验结果显示，与野生毒株相比，该突变毒株的增殖能力显著减弱，证实SR基序的磷酸化修饰对猪流行性腹泻病毒的增殖至关重要。综上，本研究明确了猪流行性腹泻病毒核衣壳蛋白的磷酸化位点及其相关调控激酶，为相关抗病毒治疗策略的开发提供了新的理论依据。