SFTSV重组假病毒的构建及Gc糖基化对病毒感染力的影响

Construction of severe fever with thrombocytopenia syndrome virus recombinant pseudoviruses and effect of Gc glycosylation on viral infectivity

目的为探究发热伴血小板减少综合征病毒(SFTSV)中Gc及其N-糖基化位点与病毒感染性的关系,构建了含有SFTSV Gc糖基化位点突变体的重组假病毒。方法利用定点突变和同源重组技术构建了SFTSV Gc及3个N-糖基化位点突变的真核表达载体pcDNA3.1(+)-Gc、pcDNA3.1(+)-Gc(N291Q)、pcDNA3.1(+)-Gc(N352Q)和pcDNA3.1(+)-Gc(N374Q)。验证其在293T细胞中成功表达后,感染VSVΔG-Fluc*G假病毒,构建4株重组假病毒并检测其对细胞感染力的影响。结果双酶切鉴定和序列测定证实成功构建真核表达载体pcDNA3.1(+)-Gc、pcDNA3.1(+)-Gc(N291Q)、pcDNA3.1(+)-Gc(N352Q)和pcDNA3.1(+)-Gc(N374Q)。间接免疫荧光和Western blot结果表明4个重组质粒均成功表达。SFTSV Gc重组假病毒对Vero细胞有感染特异性。糖基化位点突变后假病毒感染能力明显降低,且352位糖基化位点突变株感染水平最低(P<0.001、P=0.001)。结论SFTSV Gc的糖基化位点可能与病毒的感染力相关,且第352位氨基酸突变后,对病毒感染力影响最大。

Objective To explore the relationship between severe fever with thrombocytopenia syndrome virus(SFTSV)Gc and its N-glycosylation site and viral infectivity,a recombinant pseudovirus containing SFTSV Gc glycosylation site mutant was constructed.Methods The eukaryotic expression vectors pcDNA3.1(+)-GC,PCDNA3.1(+)-GC(N291Q),PCDNA3.1(+)-GC(N352Q)and PCDNA3.1(+)-GC(N374Q)were constructed by site-directed mutagenesis and homologous recombination.After their successful expression in 293T cells,we infected VSVΔG-Fluc*G pseudovirus,constructed four recombinant pseudoviruses and tested their effects on the cell force of infection.Results Double digestion identification and sequence determination confirmed the successful construction of eukaryotic expression vectors pcDNA3.1(+)-Gc,pcDNA3.1(+)-Gc(N291Q),pcDNA3.1(+)-Gc(N352Q)and pcDNA3.1(+)-Gc(N374Q).Indirect immunofluorescence and Western Blotting result indicated the successful expression of all the four recombinant plasmids.SFTSV Gc recombinant pseudoviruses are specific for infecting Vero cells.Pseudovirus infection capacity was decreased significantly after the glycosylation site mutation,and the mutant strain with the glycosylation site at position 352 had the lowest level of infectivity(P<0.001,P=0.001).Conclusions The glycosylation site of SFTSV Gc may be associated with the infectious effect of the viral infection,and the amino acid mutation at position 352 has the greatest effect on the viral infectivity.