The E301R protein of African swine fever virus functions as a sliding clamp involved in viral genome replication.
The E301R protein of African swine fever virus functions as a sliding clamp involved in viral genome replication.
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非洲猪瘟病毒 (ASFV) 是一种复杂的核质大型 DNA 病毒，可感染家猪和野猪，但人们对基因组复制过程知之甚少。此前，ASFV 的 E301R 蛋白 (pE301R) 通过将 DNA 聚合酶夹在 DNA 双链体上而被预测为增殖细胞核抗原 (PCNA) 样蛋白，但其确切结构和功能仍不清楚。在这里，pE301R的晶体结构表明它由结构相似的头域和尾域组成，并且与DNA聚合酶持续合成因子（包括滑夹和真核PCNA）具有显着的结构相似性。更具体地说，我们证明pE301R表现出多种寡聚状态（以二聚体和四聚体为主），并且四聚体与晶体堆积产生的环形同四聚体以头尾相连的方式一致。我们还表明 pE301R 与 ASFV 基因组和病毒 DNA 聚合酶 O174L 相互作用。此外，通过特定的小干扰RNA (siRNA)敲低E301R显着降低了病毒基因组复制。有趣的是，siRNA对PCNA的下调显着降低了细胞活力，而pE301R过表达则逆转了抑制作用。值得注意的是，我们证明，在转染靶向 E301R 的 siRNA 后，PCNA 的过度表达部分恢复了 ASFV 的复制。更重要的是，PCNA特异性抑制剂T2氨基醇在病毒基因组复制阶段显着抑制ASFV复制。综上所述，我们发现 pE301R 在 ASFV 基因组复制中充当滑动夹，可用作潜在的抗病毒靶点。滑夹是原核和真核细胞进化中高度保守的蛋白质。滑动夹是基因组复制所必需的，是 DNA 聚合酶的关键辅助因子。然而，病毒中的滑动钳类似物仍然很大程度上未知。我们发现 ASFV E301R 蛋白 (pE301R) 在 ASFV 复制过程中表现出滑动夹状结构和类似的功能。有趣的是，pE301R 被组装成独特的环形同源四聚体，与其他物种的滑动夹或增殖细胞核抗原 (PCNA) 不同。值得注意的是，E301R 基因是病毒生命周期所必需的，但猪 PCNA 可以部分恢复 pE301R 功能。该研究不仅强调了ASFV pE301R作为病毒滑夹类似物的功能作用，而且有助于剖析ASFV复杂的复制机制，为开发抗ASF病毒药物提供新线索。
African swine fever virus (ASFV) is a complex nucleocytoplasmic, large DNA virus that infects both domestic pigs and wild boar, but little is known about the process of genomic replication. The E301R protein (pE301R) from ASFV was previously predicted as a proliferating cell nuclear antigen (PCNA)-like protein through clamping DNA polymerase to the DNA duplex, but its exact structure and functions remain uncharacterized. Here, the crystal structure of pE301R revealed that it is composed of structurally similar head and tail domains and shares significant structural similarities to the DNA polymerase processivity factors, including sliding clamp and eukaryotic PCNA. More specifically, we demonstrated that pE301R exhibited multiple oligomeric states (with dimers and tetramers dominant), and the tetramers are consistent with the ring-shaped homotetramers in a head-to-tail manner generated by crystal packing. We also showed that pE301R interacted with the ASFV genome and viral DNA polymerase O174L. Furthermore, knockdown of E301R by specific small interfering RNAs (siRNAs) significantly decreased the virus genomic replication. Interestingly, the downregulation of PCNA by siRNAs significantly decreased the cell viability, whereas the inhibitory effect was reversed by pE301R overexpression. Notably, we demonstrated that overexpression of PCNA partially restored ASFV replication upon transfection of siRNAs targeting E301R. More importantly, T2 amino alcohol, a PCNA-specific inhibitor, markedly inhibited ASFV replication at the stage of viral genome replication. Taken together, we revealed that pE301R functions as a sliding clamp in ASFV genomic replication and can be used as a potential antiviral target. Sliding clamp is a highly conserved protein in the evolution of prokaryotic and eukaryotic cells. The sliding clamp is required for genomic replication as a critical co-factor of DNA polymerases. However, the sliding clamp analogs in viruses remain largely unknown. We found that the ASFV E301R protein (pE301R) exhibited a sliding clamp-like structure and similar functions during ASFV replication. Interestingly, pE301R is assembled into a unique ring-shaped homotetramer distinct from sliding clamps or proliferating cell nuclear antigens (PCNAs) from other species. Notably, the E301R gene is required for viral life cycle, but the pE301R function can be partially restored by the porcine PCNA. This study not only highlights the functional role of the ASFV pE301R as a viral sliding clamp analog, but also facilitates the dissection of the complex replication mechanism of ASFV, which provides novel clues for developing antivirals against ASF.
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影响因子：
5.4
作者：
Du, Xuejian;Gao, Zeng-Qiang;Zhang, Heng
通讯作者：
Zhang, Heng
影响因子：
5.6
作者：
Burnouf, DY;Olieric, V;Dumas, P
通讯作者：
Dumas, P
影响因子：
8
作者：
Matsumiya, S;Ishino, S;Morikawa, K
通讯作者：
Morikawa, K
影响因子：
64.5
作者：
KRISHNA, TSR;KONG, XP;KURIYAN, J
通讯作者：
KURIYAN, J
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC