Analysis of Cis-acting Replication Elements in Enteroviruses:The Multi-Functional Roles of the Cloverleaf Structure and the cre(2C) RNA

Abstract

Enteroviruses, such as polio- and coxsackievirus, are positive-stranded RNA viruses, and belong to the family of Picornaviruses. Positive-stranded RNA viruses follow a common mechanism to replicate their RNA genomes. First, the viral genome is transcribed into a minus-strand intermediate, which then acts as a template for the synthesis of new plus-strands. The same viral RNA-dependent RNA polymerase synthesizes both RNA strands using viral and host factors to initiate synthesis. Enteroviruses make very efficient use of their small genomes. Several cis-acting replication elements can be found throughout their genome, including one within the coding region. Most of these elements consist of RNA regions, which fold into secondary structures that can form complexes with viral and cellular proteins. Such complex formations are often required to initiate a new step in replication, and thus, function as key players in regulation of the viral life cycle. Some of the cis-acting replication elements have overlapping functions and play a role in several steps in RNA synthesis. In this thesis, two cis-acting replication elements in enteroviruses were analyzed for their roles in RNA synthesis: the cloverleaf structure in poliovirus and the cre(2C) hairpin RNA in coxsackievirus B3 (CVB3). A cloverleaf-like RNA structure formed at the 5’-end of the poliovirus plus-strand is required for negative-strand RNA synthesis but has also been implicated in positive-strand RNA synthesis. Analyzing the precise role of the cloverleaf RNA element in positive-strand RNA synthesis has been hindered by its role in negative-strand synthesis, as mutations disrupting the structure and/or functions on the cloverleaf disrupt minus-strand RNA synthesis. To overcome this limitation, we have developed a novel approach to analyze cis-acting elements with multiple roles in virus replication. Poliovirus replicons were engineered to contain two tandem cloverleaf structures to separate multiple functions. Thus, a downstream cloverleaf, which only supports minus-strand RNA synthesis, allowed the genetic analysis of a 5’-terminal cloverleaf dedicated to promote plus-strand RNA synthesis. Our results reveal that the cloverleaf structure in the plus-strand functions as a promoter for both positive- and negative-strand RNA synthesis. We could show that stem a sequences within the cloverleaf structure are essential for plus-strand RNA synthesis. Also required to initiate plus-strand RNA synthesis are the binding sites for the viral polymerase precursor 3CD and the host factor PCBP2 located within the cloverleaf structure. Furthermore, in a functional assay we could demonstrate that the viral 2C protein is directly involved in plus-strand RNA synthesis. Based on our results, we propose a new model for the initiation of positive-strand RNA synthesis in poliovirus. In the second part of this thesis, the cre(2C) RNA of coxsackievirus B3 and its role in RNA replication was analyzed. A stem-loop element located within the 2C coding region of CVB3 has been proposed to function as a cis-acting replication element. The MFOLD program was used to predict the structure and the precise location of the cre(2C) hairpin. Characterization of the cre(2C) loop showed that a proposed entero- and rhinoviral consensus sequence is also applicable to the CVB3 cre(2C) loop sequence, and that the cre(2C) element functions as a template for VPg-uridylylation in vitro. Even though previous studies of the cre(2C) in poliovirus have shown that the cre RNA is not required for initiation of negative-strand RNA synthesis, we were able to demonstrate that the CVB3 cre(2C) is required for the imitation of both, negative- and positive-strand RNA synthesis.