Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5'-Terminal Genomic Deletions.
Bottom Line: While the wt genome with the mutated CRE(2C) displays suppressed replication levels similar to those observed in a CVB3-TD strain, mutation of the CRE(2C) function in a CVB3-TD strain does not further decrease replication.In this report, we demonstrate that while CVB can replicate without a uridylylating CRE(2C), the replication rate suffers significantly.This demonstrates that VPg can prime without being specifically uridylylated and that this priming is error prone, resulting in the loss of sequence information from the 5' terminus.
Affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.Show MeSH
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Mentions: Four cis-acting replication elements (CREs) which modulate replication and translation have been identified in the HEV genome; these are at the 5′ and 3′ termini, within the ORF of the genomic positive strand, and at the 3′ end of the negative RNA strand (a replicative intermediate) (9, 10). The 5′ and 3′ ends of the genomic RNA are nontranslated regions (NTRs), and the 3′ NTR terminates in a poly(A) tail. A cloverleaf secondary structure termed domain I (Fig. 1A) is present at the 5′ terminus of the HEV genome (11, 12) and is believed to be the site of formation of the replication complex, which includes both viral and host cell proteins required for negative-strand synthesis (12–17). Binding of the viral protein 3CD to stem-loop d of domain I and the host cell protein poly(rC)-binding protein 2 (PCBP2) to stem-loop b of domain I [or the poly(C) tract in the spacer region between domain I and domain II of the internal ribosome entry site (IRES)] (Fig. 1C) has been proposed to be required for negative-strand synthesis (18–22). It has been demonstrated using a cell-free system that deletion of 4 nucleotides from stem-loop d of poliovirus RNA led to the loss of both negative-strand synthesis and the production of viral proteins (17), perhaps due to decreased binding or a loss of binding of 3CD. Further studies demonstrated that, with domain I left intact, deletion of portions or all of the IRES from poliovirus RNA did not affect the synthesis of either negative- or positive-strand genomic RNA in a cell-free system (23). These findings indicate that domain I but not the IRES of the 5′ NTR is crucial for the efficient replication of viral RNA.
Affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.