Structural dynamics of a viral genomic RNA upon host protein binding.

摘要

Coxsackieviruses, or CVBs, are responsible for a significant proportion of clinical myocarditis cases attributed to enterovirus infections. In addition to myocarditis, pancreatitis and possibly type I diabetes are caused by the Coxsackievirus serotype 3, or CVB3. CVB3 is a non-enveloped enterovirus of the Picornaviridae family. Its single-stranded, positive sense RNA genome is 7400 nucleotides long, and includes a 5' non-translated region (5'NTR), a single open reading frame, and a 3'NTR with a poly (A) tail. The local RNA structures within the CVB3 genome and particularly the internal ribosome entry site (IRES) within the 5'NTR are determinants for viral virulence. In fact, the IRES mediates cap-independent translation of the single open reading frame into a long polyprotein, which is then cleaved by viral proteases to produce mature viral proteins. The critical importance of the IRES relies on its ability to interact with viral and cellular proteins. IRES-transacting factors like the host poly (rC) binding protein 2 (or PCBP2) enhance 5'NTR activity. PCBP2 is known to regulate cellular mRNA stability and translation, but its commitment to the picornavirus life cycle depends on its affinity to C-rich regions of the 5'NTR resulting in translation enhancement and initiation of genome replication.;The goal of this research was to provide evidence of interactions of PCBP2 with domain I and domain IV of the CVB3 5'NTR and to investigate potential structural dynamics within the 5'NTR upon PCBP2 binding. Our electrophoresis mobility shift assays (EMSA) using single RNA domains confirmed interaction between PCBP2 and domain IV, but not domain I. However, our structural studies using dimethylsylfate (DMS) modification within the context of the entire 5'NTR showed protection within domain IV as well as specific alterations of RNA secondary structures in domain I. Our results suggest a model in which simultaneous protection of RNA domains by PCBP2 trigger dynamic changes in 5'NTR structure.