Zwart Lizahn, Potgieter Christiaan A, Clift Sarah J, van Staden Vida
Department of Genetics, University of Pretoria, Pretoria, South Africa.
Deltamune (Pty) Ltd, Lyttelton, Centurion, South Africa; Department of Biochemistry, Centre for Human Metabonomics, North-West University, Potchefstroom, South Africa.
PLoS One. 2015 Apr 27;10(4):e0124281. doi: 10.1371/journal.pone.0124281. eCollection 2015.
African horse sickness is a serious equid disease caused by the orbivirus African horse sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding seven structural and three non-structural proteins. Recently, an additional protein was predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9 sequences revealed the existence of two main types of AHSV NS4, designated NS4-I and NS4-II, with different lengths and amino acid sequences. The AHSV NS4 coding sequences were in the +1 reading frame relative to that of VP6. Both types of AHSV NS4 were expressed in cultured mammalian cells, with sizes close to the predicted 17-20 kDa. Fluorescence microscopy of these cells revealed a dual cytoplasmic and nuclear, but not nucleolar, distribution that was very similar for NS4-I and NS4-II. Immunohistochemistry on heart, spleen, and lung tissues from AHSV-infected horses showed that NS4 occurs in microvascular endothelial cells and mononuclear phagocytes in all of these tissues, localising to the both the cytoplasm and the nucleus. Interestingly, NS4 was also detected in stellate-shaped dendritic macrophage-like cells with long cytoplasmic processes in the red pulp of the spleen. Finally, nucleic acid protection assays using bacterially expressed recombinant AHSV NS4 showed that both types of AHSV NS4 bind dsDNA, but not dsRNA. Further studies will be required to determine the exact function of AHSV NS4 during viral replication.
非洲马瘟是由环状病毒非洲马瘟病毒（AHSV）引起的一种严重的马属动物疾病。该病毒有10个双链RNA基因组片段，编码7种结构蛋白和3种非结构蛋白。最近，预测大多数环状病毒的基因组片段9（Seg-9）除了编码VP6外，还编码一种额外的蛋白质。这一点后来在蓝舌病毒和大岛病毒中得到了证实，这种非结构蛋白被命名为NS4。在本研究中，对AHSV Seg-9序列的计算机分析揭示了两种主要类型的AHSV NS4的存在，分别命名为NS4-I和NS4-II，它们具有不同的长度和氨基酸序列。AHSV NS4编码序列相对于VP6的编码序列处于+1阅读框。两种类型的AHSV NS4都在培养的哺乳动物细胞中表达，大小接近预测的17 - 20 kDa。对这些细胞进行荧光显微镜检查发现，NS4-I和NS4-II在细胞质和细胞核中都有分布，但不在核仁中，二者分布非常相似。对AHSV感染马匹的心脏、脾脏和肺组织进行免疫组织化学分析表明，NS4存在于所有这些组织的微血管内皮细胞和单核吞噬细胞中，定位于细胞质和细胞核。有趣的是，在脾脏红髓中具有长细胞质突起的星状树突状巨噬细胞样细胞中也检测到了NS4。最后，使用细菌表达的重组AHSV NS4进行核酸保护试验表明，两种类型的AHSV NS4都能结合双链DNA，但不能结合双链RNA。需要进一步研究来确定AHSV NS4在病毒复制过程中的具体功能。
