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

Abstract

We have previously shown that the RNA polymerase 3D ^pol of human rhinovirus 2 (HRV2) catalyzes the covalent linkage of UMP to the terminal protein (VPg) using poly(A) as a template (K. Gerber, E. Wimmer, and A. V. Paul, J. Virol. 75:10969–10978, 2001). The products of this in vitro reaction are VPgpU, VPgpUpU, and VPg-poly(U), the 5′ end of minus-strand RNA. In the present study we used an assay system developed for poliovirus 3D ^pol (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74: 10359–10370, 2000) to search for a viral sequence or structure in HRV2 RNA that would 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, yielding VPgpU and VPgpUpU. The in vitro reaction is strongly stimulated by the addition of purified HRV2 3CD ^pro . Our analyses suggest that HRV2 3D ^pol uses a “slide-back” mechanism during synthesis of the VPg-linked precursors. The corresponding cis- replicating RNA elements in the 2C ^ATPase coding region of poliovirus type 1 Mahoney (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 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 uridylylation reaction.