Objective      To screen host proteins interacting with enterovirus 71 (EV71) 3D RNA polymerase by tandem affinity purification (TAP).       Methods      The full-length 3D RNA polymerase gene was amplified from the whole genome of EV71 BrCr strain by RT-PCR

and then the fragment was inserted into vector pcDNA3.0-Flag-HA to construct pcDNA3.0-3D-Flag-HA eukaryotic expression vector. The recombinant plasmid was transferred into RD cells

and after 48 h the RD cells were collected. Western blotting was applied to detect the expression of 3D RNA polymerase in the RD cells. TAP and liquid chromatography/mass spectrometry (LC/MS) were performed to screen 3D RNA polymerase-interacting host proteins. Co-immunoprecipitation (Co-IP) was applied to confirm the interaction between candidate proteins (such as cyclin G1) and 3D RNA polymerase.       Results      Recombinant plasmid pcDNA3.0-3D-Flag-HA was successfully constructed and 3D RNA polymerase was expressed in the RD cells. A number of 3D RNA polymerase-interacting proteins including LATS2

Nek

APC5

Cyclin G1 and PI3K that were involved in cell apoptosis and regulation of cell cycle were identified by TAP purification and LC/MS. The interaction between Cyclin G1 and 3D RNA polymerase was confirmed by Co-IP.       Conclusion      EV71 3D RNA polymerase may interact with host proteins to induce cell apoptosis and cell cycle changes.