许多正链RNA病毒的5'非翻译区(5'UTR)含有功能性调节序列。这里,我们证明猪繁殖与呼吸综合征病毒(PRRSV),动脉炎病毒的成员,在其5'UTR中拥有小的上游开放阅读框(uORFs)。生物信息学分析表明,该特征在PRRSV毒株和动脉炎病毒科中相对较好地保守。我们还确认了一个uORF,即,在PRRSV株JXwn06中的uORF2,其具有翻译活性并且对通过体外报告物测定证明的初级ORF的表达发挥抑制作用。我们通过反向遗传学测试了其重要性,方法是将点突变引入PRRSV感染性cDNA克隆中以使uORF2的起始密码子失活。回收的突变病毒Mut2令人惊讶地复制到与野生型病毒(WT)相同的水平,但诱导了更高水平的炎症细胞因子(例如,TNF-α,IL-1β,和IL-6)在体外和动物实验中,与更严重的肺损伤和更高的死亡率相关。与此相符,转染细胞中uORF2的过表达显著抑制poly(I:C)诱导的炎性细胞因子的表达。一起,我们的数据支持uORF2编码小说的想法,PRRSV毒力的功能调节剂,尽管它的规模很短。
目的:PRRSV仍然是世界养猪业面临的重大挑战,但是我们对它的生物学和发病机制仍然知之甚少。这里,我们提供的证据表明,PRRSV株JXwn06的5'UTR具有功能性uORF,该uORF具有编码能力并调节炎症的诱导,如体外测定和动物实验所证明。这些发现揭示了一种调节细胞炎症的新的病毒因子,并提供了对PRRSV发病机理的理解。
The 5\' untranslated region (5\'UTR) of many positive-stranded RNA viruses contain functional regulatory sequences. Here, we show that the porcine reproductive and respiratory syndrome virus (PRRSV), a member of arteriviruses, harbors small upstream open reading frames (uORFs) in its 5\'UTR. Bioinformatics analysis shows that this feature is relatively well conserved among PRRSV strains and Arteriviridae. We also identified a
uORF, namely uORF2, in the PRRSV strain JXwn06, that possesses translational activity and exerts a suppressive effect on the expression of the primary ORF evidenced by in vitro reporter assays. We tested its importance via reverse genetics by introducing a point mutation into the PRRSV infectious cDNA clone to inactivate the start codon of uORF2. The recovered mutant virus Mut2 surprisingly replicated to the same level as the wild-type virus (WT), but induced a higher level of inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) both in vitro and in animal experiments, correlating well with more severe lung injury and higher death rate. In line with this, over-expression of uORF2 in transfected cells significantly inhibited poly(I:C)-induced expression of inflammatory cytokines. Together, our data support the idea that uORF2 encodes a novel, functional regulator of PRRSV virulence despite of its short size.
OBJECTIVE: PRRSV has remained a major challenge to the world swine industry, but we still do not know much about its biology and pathogenesis. Here, we provide evidence to show that the 5\'UTR of PRRSV strain JXwn06 harbors a functional
uORF that has the coding capacity and regulates induction of inflammation as demonstrated by in vitro assays and animal experiment. The findings reveal a novel viral factor that regulates cellular inflammation and provide insight into the understanding of PRRSV pathogenesis.