鸭坦布苏病毒(DTMUV)属于黄病毒科,主要感染鸭。鸭Tembusu病毒基因组编码一种经过切割产生10种蛋白质的多蛋白。其中,NS4B,最大的跨膜蛋白,在病毒生命周期中起着至关重要的作用。在这项研究中,我们调查了NS4B的定位,发现它位于内质网,它与DTMUVdsRNA共定位。随后,我们证实了NS4B的5个不同的跨膜结构域,并发现只有其跨膜结构域3(TMD3)可以穿过ER膜。然后在DTMUV复制子和感染性克隆的NS4BTMD3的保守氨基酸中引入突变。结果表明,V111G,V117G,和I118G突变增强病毒RNA复制,而Q104A,T106A,A113L,M116A,H120A,Y121A,和A122G突变减少病毒复制。在BHK21细胞中拯救并研究了具有这些突变的重组病毒。研究结果表明,A113L和H120A突变导致比野生型菌株更高的病毒滴度,而Q104A,T106A,V111G,V117G,和Y121A突变减弱病毒增殖。此外,H120A,M116A,和A122G突变增强病毒增殖。此外,Q104A,T106A,V111G,M116A,V117G,Y121A,A122G突变体对10d鸭胚的病毒毒力降低。动物实验进一步表明,与感染后5天的WT组相比,所有突变病毒导致脾脏中更低的基因组拷贝数。我们的数据提供了对DTMUVNS4B拓扑模型的见解,强调NS4BTMD3在病毒复制和增殖中的重要作用。
Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation.