Kanade Gayatri D, Pingale Kunal D, Karpe Yogesh A
Agharkar Research Institute, Nanobioscience Group, Pune, India.
Savitribai Phule Pune University, Pune, India.
J Virol. 2018 Feb 26;92(6). doi: 10.1128/JVI.01853-17. Print 2018 Mar 15.
Hepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstrated processing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV. Hepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.
戊型肝炎病毒（HEV）是一种具有临床重要性的正链RNA病毒。HEV的开放阅读框1（ORF1）编码一个由1693个氨基酸组成的非结构多聚蛋白。目前尚不清楚ORF1多聚蛋白（pORF1）是否会被加工成不同的酶结构域。许多研究人员试图了解pORF1的加工机制。然而，这些研究得出了各种不同的结果，始终未能令人信服地确定pORF1的加工机制。在本研究中，我们证明了凝血酶和凝血因子Xa在pORF1加工过程中可能发挥的作用。我们观察到HEV的pORF1多聚蛋白具有凝血酶和凝血因子Xa的保守切割位点。通过反向遗传学方法，我们证明了在凝血酶或凝血因子Xa切割位点发生突变的HEV复制子在细胞培养中不能有效复制。此外，当我们将重组pORF1片段与凝血酶一起孵育时，我们观察到了pORF1多聚蛋白的加工过程。用丝氨酸蛋白酶抑制剂处理肝细胞系以及通过小干扰RNA（siRNA）敲低凝血酶和凝血因子Xa，均导致HEV复制显著减少。凝血酶和凝血因子Xa在血液凝固中的作用已得到充分研究。据信这两种蛋白在血浆中均以活性形式存在。有趣的是，在本报告中，我们证明了肝细胞系中存在具有生物活性的凝血酶和凝血因子Xa。结果表明，凝血因子Xa和凝血酶对于HEV的复制至关重要，可能参与了HEV的pORF1多聚蛋白加工过程。戊型肝炎病毒（HEV）在人类中会引发一种称为肝炎的肝脏疾病，在成年人中大多为急性自限性感染。在发展中国家，感染HEV的孕妇死亡率高达约30%。由于迄今为止所获得的研究结果存在差异，关于HEV ORF1多聚蛋白的加工尚无令人信服的观点。HEV的pORF1具有两个宿主细胞丝氨酸蛋白酶（凝血酶和凝血因子Xa）的切割位点，这些位点在HEV各基因型中保守。本研究首次证明，HEV pORF1上的凝血酶和凝血因子Xa切割位点对于HEV复制是必不可少的。上述丝氨酸蛋白酶的细胞内生化活性对于HEV在细胞培养中的有效复制也至关重要，并且必定参与了pORF1的加工过程。本研究揭示了凝血因子在细胞内病毒复制方面的存在及作用。
