Wall Gayle V, Wright Isabella M, Barnardo Carin, Erasmus Baltus J, van Staden Vida, Potgieter A Christiaan
Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria, 0002, South Africa.
Deltamune (Pty) Ltd, Moraine House - The Braes, 193 Bryanston Drive, Bryanston, Gauteng, 2191, South Africa.
Virus Res. 2021 Jun;298:198407. doi: 10.1016/j.virusres.2021.198407. Epub 2021 Apr 1.
African horse sickness virus (AHSV) non-structural protein NS4 is a nucleocytoplasmic protein that is expressed in the heart, lung, and spleen of infected horses, binds dsDNA, and colocalizes with promyelocytic leukemia nuclear bodies (PML-NBs). The aim of this study was to investigate the role of AHSV NS4 in viral replication, virulence and the host immune response. Using a reverse genetics-derived virulent strain of AHSV-5 and NS4 deletion mutants, we showed that knockdown of NS4 expression has no impact in cell culture, but results in virus attenuation in infected horses. RNA sequencing (RNA-seq) was used to investigate the transcriptional response in these horses, to see how the lack of NS4 mediates the transition of the virus from virulent to attenuated. The presence of NS4 was shown to result in a 24 hour (h) delay in the transcriptional activation of several immune system processes compared to when the protein was absent. Included in these processes were the RIG-I-like, Toll-like receptor, and JAK-STAT signaling pathways, which are key pathways involved in innate immunity and the antiviral response. Thus, it was shown that AHSV NS4 suppresses the host innate immune transcriptional response in the early stages of the infection cycle. We investigated whether AHSV NS4 affects the innate immune response by impacting the JAK-STAT signaling pathway specifically. Using confocal laser scanning microscopy (CLSM) we showed that AHSV NS4 disrupts JAK-STAT signaling by interfering with the phosphorylation and/or translocation of STAT1 and pSTAT1 into the nucleus. Overall, these results showed that AHSV NS4 is a key virulence factor in horses and allows AHSV to overcome host antiviral responses in order to promote viral replication and spread.
非洲马瘟病毒（AHSV）的非结构蛋白NS4是一种核质蛋白，在受感染马匹的心脏、肺和脾脏中表达，可结合双链DNA，并与早幼粒细胞白血病核体（PML-NBs）共定位。本研究的目的是调查AHSV NS4在病毒复制、毒力和宿主免疫反应中的作用。使用反向遗传学衍生的AHSV-5强毒株和NS4缺失突变体，我们发现敲低NS4表达对细胞培养没有影响，但会导致感染马匹的病毒减毒。RNA测序（RNA-seq）用于研究这些马匹的转录反应，以了解NS4的缺失如何介导病毒从强毒株向减毒株的转变。与不存在该蛋白时相比，NS4的存在导致几个免疫系统过程的转录激活延迟24小时（h）。这些过程包括RIG-I样、Toll样受体和JAK-STAT信号通路，它们是参与先天免疫和抗病毒反应的关键通路。因此，研究表明AHSV NS4在感染周期的早期阶段抑制宿主先天免疫转录反应。我们专门研究了AHSV NS4是否通过影响JAK-STAT信号通路来影响先天免疫反应。使用共聚焦激光扫描显微镜（CLSM），我们发现AHSV NS4通过干扰STAT1和pSTAT1的磷酸化和/或向细胞核的转运来破坏JAK-STAT信号通路。总体而言，这些结果表明AHSV NS4是马匹中的关键毒力因子，使AHSV能够克服宿主的抗病毒反应，从而促进病毒的复制和传播。
