Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: Identification of a cis-replicating element in the coding sequence of 2A(pro)

We have previously shown that the RNA polymerase 3D(pol) of human rhinoviru s 2 (HRV2) catalyzes the covalent linkage of UMP to the terminal protein (V Pg) using poly(A) as a template (K. Gerber, E. Wimmer, and A. V. Paul, J. V irol. 75:10969-10978, 2001). The products of this in vitro reaction are VPg pU, VPgpUpU, and VPg-poly(U), the 5' end of minus-strand RNA. In the presen t study we used an assay system developed for poliovirus 3D(pol) (A. V. Pau l, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74: 10359 -10370, 2000) to search for viral sequence or structure in HRV2 RNA that wo uld provide specificity to this reaction. We now show that a small hairpin in HRV2 RNA [cre(2A)], located in the coding sequence of 2A(pro), serves as the primary template for HRV2 3D(pol) in the uridylylation of HRV2 VPg, yi elding VPgpU and VPgpUpU. The in vitro reaction is strongly stimulated by t he addition of purified HRV2 3CD(pro). Our analyses suggest that HRV2 3D(po l) uses a "slide-back" mechanism during synthesis of the VPg-linked precurs ors. The corresponding cis- replicating RNA elements in the 2C(ATPase) codi ng region of poliovirus type 1 Mahoney (I. Goodfellow, Y. Chaudhry, A. Rich ardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 7 4:4590-4600, 2000) and VP1 of HRV14 (K. L. McKnight and S. M. Lemon, RNA 4: 1569-1584, 1998) can be functionally exchanged in the assay with cre(2A) of HRV2. Mutations of either the first or the second A in the conserved A(1)A (2)A(3)CA sequence in the loop of HRV2 cre(2A) abolished both viral growth and the RNA's ability to serve as a template in the in vitro VPg uridylylat ion reaction.