A temperature-sensitive defect of enterovirus 70 is located at the uridylylation of the genome-linked protein VPg in vitro.

The temperature-sensitive phenotype of enterovirus 70 (EV70) was examined by use of an in-vitro RNA replication system derived from a membrane fraction (crude replication complex, CRC) of EV70-infected HeLa cells. This system was capable of synthesizing the nucleotidyl proteins VPg-pU and VPg-pUpU. Formation of these nucleotidyl proteins was completely abolished when the in-vitro reaction was performed at the nonpermissive temperature for virus replication. Considering our previous observation that the defective stage of the temperature-sensitive growth of EV70 resides in the initiation step of RNA transcription in vivo, it is most likely that the lack of uridylylation of VPg at the restricted temperature in vitro is directly involved in the temperature-sensitive defect of virus growth in vivo.