Affiliation:
1. Department of Microbiology
2. Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262
Abstract
ABSTRACT
The
cis
-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU
OH
, the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU
OH
, however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU
OH
synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis.
trans
-replication experiments demonstrated that the CRE and VPgpUpU
OH
were not required in
cis
or in
trans
for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU
OH
in the asymmetric replication of poliovirus RNA.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
Cited by
115 articles.
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