Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg

The first step in the replication of the plus-stranded poliovirus RNA is th e synthesis of a complementary minus strand. This process is initiated by t he covalent attachment of UMP to the terminal protein VPg, yielding VPgpU a nd VPgpUpU. We have previously shown that these products can be made in vit ro in a reaction that requires only synthetic VPg, UTP, poly(A), purified p oliovirus RNA polymerase 3D(pol), and Mg2+ (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since such a poly(A)-de pendent process cannot confer sufficient specificity to poliovirus RNA repl ication, we have developed a new assay to search for a viral RNA template i n conjunction with viral or cellular factors that could provide this functi on. We have now discovered a small RNA hairpin in the coding region of prot ein 2C as the site in PV1(M) RNA that is used as the primary template for t he in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of min us strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Mere dith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 20 00). The uridylylation reaction either with transcripts of cre(2C) RNA or,v ith full-length PV1(M) RNA as the template is strongly stimulated by the ad dition of purified viral protein 3CD(pro). Deletion of the cre(2C) RNA sequ ences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C ) can be functionally exchanged in the assay. The mechanism by which the VP gpUpU precursor, made specifically on the cre(2C) template, might be transf erred to the site where it serves as primer for poliovirus RNA synthesis, r emains to be determined.