Brinton Margo A, Di Han, Vatter Heather A
Georgia State University, Atlanta, GA, USA.
Georgia State University, Atlanta, GA, USA.
Virus Res. 2015 Apr 16;202:112-9. doi: 10.1016/j.virusres.2014.11.024. Epub 2014 Nov 29.
The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arteriviridae in encoding three papain-like one proteases (PLP1α, PLP1β and PLP1γ) at the 5' end and two adjacent sets of four minor structural proteins at the 3' end. The catalytic Cys and His residues and cleavage sites for each of the SHFV PLP1s were predicted and their functionality was tested in in vitro transcription/translation reactions done with wildtype or mutant polyprotein constructs. Mass spectrometry analyses of selected autoproteolytic products confirmed cleavage site locations. The catalytic Cys of PLP1α is unusual in being adjacent to an Ala instead of a Typ. PLP1γ cleaves at both downstream and upstream sites. Intermediate precursor and alternative cleavage products were detected in the in vitro transcription/translation reactions but only the three mature nsp1 proteins were detected in SHFV-infected MA104 cell lysates with SHFV nsp1 protein-specific antibodies. The duplicated sets of SHFV minor structural proteins were predicted to be functionally redundant. A stable, full-length, infectious SHFV-LVR cDNA clone was constructed and a set of mutant infectious clones was generated each with the start codon of one of the minor structural proteins mutated. All eight of the minor structural proteins were found to be required for production of infectious extracellular virus. SHFV causes a fatal hemorrhagic fever in macaques but asymptomatic, persistent infections in natural hosts such as baboons. SHFV infections were compared in macrophages and myeloid dendritic cells from baboons and macaques. Virus yields were higher from macaque cells than from baboon cells. Macrophage cultures from the two types of animals differed dramatically in the percentage of cells infected. In contrast, similar percentages of myeloid dendritic cells were infected but virus replication was efficient in the macaque cells but inefficient in the baboon cells. SHFV infection induced the production of pro-inflammatory cytokines, including IL-1β, IL-6, IL-12/23(p40), TNF-α and MIP-1α, in macaque cells but not baboon cells.
猴出血热病毒（SHFV）基因组与动脉炎病毒科其他成员的基因组不同，它在5'端编码三种类木瓜蛋白酶（PLP1α、PLP1β和PLP1γ），在3'端编码两组相邻的四个次要结构蛋白。预测了每个SHFV PLP1的催化半胱氨酸和组氨酸残基以及切割位点，并在使用野生型或突变多蛋白构建体进行的体外转录/翻译反应中测试了它们的功能。对选定的自蛋白水解产物进行质谱分析，确认了切割位点的位置。PLP1α的催化半胱氨酸不寻常之处在于它与丙氨酸相邻，而不是与苏氨酸相邻。PLP1γ在下游和上游位点都有切割作用。在体外转录/翻译反应中检测到中间前体和替代切割产物，但在用SHFV nsp1蛋白特异性抗体检测的SHFV感染的MA104细胞裂解物中，仅检测到三种成熟的nsp1蛋白。预测SHFV次要结构蛋白的重复组在功能上是冗余的。构建了一个稳定的、全长的、有感染性的SHFV-LVR cDNA克隆，并产生了一组突变感染性克隆，每个克隆的一个次要结构蛋白的起始密码子发生了突变。发现所有八个次要结构蛋白都是产生有感染性的细胞外病毒所必需的。SHFV在猕猴中引起致命的出血热，但在狒狒等天然宿主中引起无症状的持续性感染。比较了来自狒狒和猕猴的巨噬细胞和髓样树突状细胞中的SHFV感染情况。猕猴细胞的病毒产量高于狒狒细胞。两种动物的巨噬细胞培养物在感染细胞的百分比上有很大差异。相比之下，髓样树突状细胞的感染百分比相似，但病毒在猕猴细胞中复制有效，而在狒狒细胞中复制效率低下。SHFV感染在猕猴细胞中诱导了促炎细胞因子的产生，包括IL-1β、IL-6、IL-12/23(p40)、TNF-α和MIP-1α，但在狒狒细胞中没有。
