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The non-structure protein （NSP） 12 of feline infectious peritonitis virus （FIPV） serves as the catalytic core component of the RNA-dependent RNA polymerase （RdRp）. However， the molecular mechanisms regulating its interactions with NSP7 and NSP8， as well as the enzymatic activity and function of the RdRp complex， remain to be fully elucidated. This study aims to investigate the molecular characteristics of NSP12， explore its interaction with NSP7 and NSP8， and verify the enzymatic activity of the RdRp complex. Firstly， the physicochemical properties and structural features of NSP12 were predicted by bioinformatics analysis. To investigate the interaction between NSP7， NSP8 and NSP12 in the FIPV RdRp complex， pcDNA3.1-Flag-NSP7， pcDNA3.1-HA-NSP8 and pcDNA3.1-Myc-NSP12 plasmids were constructed and expressed in 293T cells. The protein interactions and co-localization were detected using co-immunoprecipitation （Co-IP）， pull-down， and indirect immunofluorescence assay （IFA）. Secondly， the NSP7， NSP8 and NSP12 genes of FIPV were amplified by PCR and homologously recombined into the pET-30α vector to construct prokaryotic expression recombinant plasmids. The induced expression conditions were further optimized， and the proteins were purified by nickel column affinity chromatography and concentrated using an ultrafiltration tube to obtain the RdRp complex. Finally， an in vitro RNA elongation system for FIPV RdRp was established， and the enzymatic activity of the RdRp complexes obtained above was tested using the gel method. Bioinformatics analysis showed that NSP12 was a stable hydrophilic protein， containing 43.51% α helices， 45.02% random coils and 90 potential phosphorylation sites. The Co-IP， pull-down， and IFA assays confirmed the interaction and co-localization of NSP7， NSP8 and NSP12 in 293T cells. NSP7 and NSP8 were expressed as inclusion bodies， with molecular masses of 10 and 23 ku， respectively. In contrast， NSP12 was expressed as a soluble protein， with a molecular mass of 105 ku. The results of the enzyme activity assay demonstrated that the RdRp complex exhibited the capacity to extend the RNA template strand. Conversely， NSP12 alone exhibited no such activity. In this study， the FIPV RdRp complex was purified by prokaryotic expression and an enzyme activity detection system was established in vitro. It was confirmed for the first time that NSP12 from the FIPV-DF2 strain requires the formation of a complex with NSP7 and NSP8 to exert its RdRp function. This study lays the foundation for further exploring the biological function of FIPV RdRp and developing antiviral drugs targeting RdRp.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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