UBC Theses and Dissertations
Specific protein interactions with the 5’- and 3’-untranslated regions of coxsackievirus B3 RNA Cheung, Paul Kim-ming
In recent years, studies of the molecular pathogenesis of coxsackievirus B3 (CVB3) have revealed that the viral 5' and 3' untranslated regions (UTRs) contain determinants of viral tissue tropism, translation efficiency, and cardiovirulence. However, the mechanism that links the UTRs to these phenotypic observations remains unknown. Thus, the study of protein-UTR interactions is important to understand the underlying mechanism of functions mediated by the CVB3 UTRs. My dissertation addresses this subject by the identification and characterization of cellular proteins that bind to the CVB3 UTRs. To achieve these goals, I sub-cloned the CVB3 5' and 3'-UTR by a PCR-based strategy. The 5'-UTR sequences of nt 1-209, 210-529 and 530-630 were cloned in consideration of their respective structural and functional significance. At the same time, the 3'-UTR wild type sequence of nt 7299-7399 [+ poly(A) tail] and five mutated 3'- UTR clones were generated. Mutations in these 3'-UTR clones were previously reported to cause tertiary (but not secondary) structural changes in the 3'-UTR, leading to specific inhibition of viral replication. By using radiolabeled RNA transcripts of these UTR sequences in gel mobility shift and competitive UV cross-linking assays, fifteen specific CVB3 5'-UTR-binding proteins in the HeLa cell were identified. The molecular weight of six important 5'-UTRbinding proteins resembled those of the eukaryotic translation initiation factors (eIF4A, 4B, 4G), the death associated protein-5, La autoantigen and the polypyrimidine tract binding proteins. Using the same method, protein interactions with the 3'-UTR and its various mutants were also investigated. Two small proteins (22 and 24 kDa) that bound specifically to the mutated 3'- UTRs were observed. As these corresponding mutations were previously shown to inhibit viral replication, a functional role of these two protein-3'-UTR interactions was inferred. Also, the poly(A) tail of the 3'-UTR was found to be important in mediating HeLa cell protein interactions. As the poly(A) tail can interact with the 3'-UTR sequence to maintain a stable secondary RNA structure, my data suggests an important role of the stem-loop RNA structures in mediating protein-3'-UTR interactions. In the A/J mouse model, by using in situ hybridization and UV cross-linking assays, the correlation between CVB3 tissue tropism and protein-5'-UTR interaction was investigated. Among the various UTR-binding proteins in different tissues, a 28 kDa kidney protein was found to bind to the antisense 5'-UTR sequence of nt 210-529 and correlated well with the low viral infectivity in the kidneys, thereby suggesting a possible inhibitory function of this 28 kDa protein. Moreover, a specific 5'- and 3'-UTR-binding protein that shares the same molecular weight (52 kDa) with the La autoantigen, was chosen for further investigation. Using recombinant GST-La fusion protein from purified from E. coli, interactions between the La protein and the 5' or 3'-UTR radiolabeled probes was verified. Specificity of interaction was determined from competitive UV cross-linking experiments. It was observed that the La protein had the highest affinity towards nt 210-529, followed by the nt 530-630 (internal ribosomal entry site, IRES) and then nt 1-209. The lack of primary sequence consensus among these 5'-UTRs suggests that the La protein recognizes secondary RNA structures during interaction. This is also supported by the observation that the La protein interacted with both the wild type and mutated 3'-UTR RNAs, both of which share identical secondary structures. Analysis of the data suggests that each La protein may bind either one of the 5' and 3'-UTR at a time.
Item Citations and Data