Young Lucy B, Melian Ezequiel Balmori, Setoh Yin Xiang, Young Paul R, Khromykh Alexander A
Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia.
J Gen Virol. 2015 May;96(Pt 5):1042-1054. doi: 10.1099/vir.0.000053. Epub 2015 Jan 22.
West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS1', a C-terminal extended product of NS1 generated as the result of a -1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS1', resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS1', or a combination of both, remains undetermined. Here, we showed that WNV NS1' formed a unique subpopulation of heat- and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS1'-expressing plasmid showed that stability of NS1' dimers was linked to the penultimate 10 aa. To examine the role of NS1' heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1' to create a WNV producing a truncated version of NS1' lacking the last 20 aa, but not affecting the PRF. NS1' protein produced by this mutant virus was secreted more efficiently than WT NS1', indicating that the sequence of the last 20 aa of NS1' was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNVKUN, suggesting that full-length NS1' was not essential for virus replication in vitro and for virulence in mice.
西尼罗河病毒（WNV）是一种由蚊子传播的黄病毒，是美国虫媒病毒性脑炎的主要病因。与日本脑炎病毒血清群的其他成员一样，WNV会产生一种额外的非结构蛋白NS1'，它是NS1的C末端延伸产物，由 -1程序性核糖体移码（PRF）产生。我们之前已经表明，消除PRF从而消除NS1'的突变会导致神经侵袭性降低。然而，这是由PRF事件本身引起的，还是由缺乏PRF产物NS1'引起的，或者是两者共同作用的结果，仍未确定。在这里，我们表明WNV NS1'形成了一个独特的热稳定和低pH稳定二聚体亚群。使用表达NS1'的质粒进行的C末端截短和突变分析表明，NS1'二聚体的稳定性与倒数第二个10个氨基酸有关。为了研究NS1'热稳定二聚体在病毒复制和致病性中的作用，在NS1'中引入了一个终止密码子突变，以创建一种产生截短版NS1'的WNV，该截短版NS1'缺少最后20个氨基酸，但不影响PRF。这种突变病毒产生的NS1'蛋白比野生型NS1'更有效地分泌，这表明NS1'最后20个氨基酸的序列负责其细胞内保留。对这种突变体的进一步分析表明，外周感染后在细胞中的生长动力学和在断奶小鼠中的毒力与野生型WNV KUN相似，这表明全长NS1'对于体外病毒复制和小鼠毒力不是必需的。
