Pflag cmv 2

The detailed construction of R1 replicon has been described previously (Su et al., 2020 (link)). Briefly, for R1 3D^D330A, in which Asp330 was mutated to an Ala to disrupt the enzymatic activity, was constructed by PCR-based site-directed mutagenesis using the R1 replicon as a template (Su et al., 2020 (link)). The monocistronic reporter plasmid (IRES-Luc), which contains EV-A71 IRES at the 5′end and drives the translation of the luciferase protein, was described previously (Su et al., 2018 (link)). To construct pFlag-CMV2-HSPA6, HSPA6 gene was amplified from the cDNA of EV-A71 infected RD cells by nested PCR and then cloned into pFlag-CMV2 (Sigma-Aldrich) at NotI and KpnI sites to generate pFlag-HSPA6. A wobble HSPA6 mutation (nucleotides 7–9 GCC→GCA), which resists the action of sgRNA used in knockout cells, was generated by site-direct mutagenesis. HCV IRES-Luc was previously described (Su et al., 2018 (link)). CV-A16 IRES-Luc and Echo 9 IRES-Luc were gifts from Dr. Szu-Hao Kung (National Yang-Ming University, Taiwan) (Hou et al., 2016 (link)). EMCV IRES-Luc was generated by inserting firefly Luc gene into the pTM1 plasmid (Addgene).

Full‐length cDNAs encoding dynAPa (NCBI accession number: NM_173629.1), dynAPb (NCBI accession number: NM_001307955.1), and dynAPc (NCBI accession number: XM_011525924.2) were subcloned into the pFLAG‐CMV2‐Bsd vector, a derivative of pFLAG‐CMV‐2 (Sigma‐Aldrich) carrying the blasticidin S deaminase gene, yielding plasmids expressing N‐terminally Flag‐tagged dynAPa–c. For retrovirus‐mediated expression, untagged dynAPa–c cDNAs were incorporated into the EcoRI and XhoI sites of the pMY‐IRES‐EGFP vector as described previously [9 (link)]. The resulting plasmids were introduced into Plat‐E cells using FuGENE 6 transfection reagent (Promega Co., Madison, WI, USA) according to the manufacturer's instructions. After 48 h, virus‐containing supernatants were filtered through 0.45 μm cellulose acetate filters and supplemented with 8 μg·mL⁻¹ polybrene (Sigma‐Aldrich). NIH3T3 cells were then incubated overnight with the virus/polybrene‐containing supernatants. After infection, the medium was replaced with fresh medium. The expression, subcellular localization, and in vitro cell transformation ability were assessed in the resultant cells. Plasmids expressing N‐terminally Flag‐tagged dynAPa–c were transiently transfected into KMST‐6 cells using FuGENE HD (Promega), and subcellular localization of dynAPa–c was analyzed.

The empty expression vector pRcCMV was purchased from Invitrogen (Invitrogen Life Technologies, Grand Island, NY, USA). The plasmid pRcCMV-agno which encodes BKPyV agnoprotein has been previously described [7 (link)]. Plasmid pFLAG-CMV-2 was obtained from Sigma Aldrich, while pFLAG-CMV-2 PCNA expression vector was a kind gift of Hong-Gang Wang [69 (link)]. pGST-agno has been described previously [34 (link)].