The E301R protein of African swine fever virus functions as a sliding clamp involved in viral genome replication
Su Li,
Hailiang Ge,
Yanhua Li,
Kehui Zhang,
Shaoxiong Yu,
Hongwei Cao,
Yanjin Wang,
Hao Deng,
Jiaqi Li,
Jingwen Dai,
Lian-Feng Li,
Yuzi Luo,
Yuan Sun,
Zhi Geng,
Yuhui Dong,
Heng Zhang,
Hua-Ji Qiu
Affiliations
Su Li
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Hailiang Ge
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Yanhua Li
Multidiscipline Research Center, Institute of High Energy Physics, Chinese Academy of Sciences , Beijing, China
Kehui Zhang
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Shaoxiong Yu
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Hongwei Cao
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Yanjin Wang
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Hao Deng
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Jiaqi Li
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Jingwen Dai
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Lian-Feng Li
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Yuzi Luo
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Yuan Sun
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
Zhi Geng
Multidiscipline Research Center, Institute of High Energy Physics, Chinese Academy of Sciences , Beijing, China
Yuhui Dong
Multidiscipline Research Center, Institute of High Energy Physics, Chinese Academy of Sciences , Beijing, China
Heng Zhang
Multidiscipline Research Center, Institute of High Energy Physics, Chinese Academy of Sciences , Beijing, China
Hua-Ji Qiu
State Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-Reference Laboratory, National High-Containment Facilities for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China
ABSTRACT 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. IMPORTANCE 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.
