Hepatitis C virus RNA: molecular switches mediated by long-range RNA–RNA interactions?

Abstract

Multiple conserved structural cis-acting regulatory elements have been recognized both in the coding and untranslated regions (UTRs) of the hepatitis C virus (HCV) genome. For example, the cis-element 5BSL3.2 in the HCV-coding region has been predicted to use both its apical and internal loops to interact with the X RNA in the 3′-UTR, with the IIId domain in the 5′-UTR and with the Alt sequence in the coding region. Additionally, the X RNA region uses a palindromic sequence that overlaps the sequence required for the interaction with 5BSL3.2, to dimerize with another HCV genome. The ability of the 5BSL3.2 and X RNA regions to engage in multi-interactions suggests the existence of one or more molecular RNA switches which may regulate different steps of the HCV life cycle. In this study, we used biophysical methods to characterize the essential interactions of these HCV cis-elements at the molecular level. Our results indicate that X RNA interacts with 5BSL3.2 and another X RNA molecule by adopting two different conformations and that 5BSL3.2 engages simultaneously in kissing interactions using its apical and internal loops. Based on these results, we propose a mode of action for possible molecular switches involving the HCV RNA.

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

“…These results, obtained with short RNA sequences, are in good agreement with a recent work with longer RNAs analyzed by SHAPE mapping that suggested that these interactions could occur simultaneously (Tuplin et al 2012). Together, they strongly support previous works suggesting that 5BSL3.2 could be involved in a complex pseudoknot, implicating nonexclusive, long-range interactions between the internal loop of 5BSL3.2 and the sequence centered on nucleotide 9110 and between the apical loop of 5BSL3.2 and SL2 (Diviney et al 2008;Shetty et al 2013).…”

“…Binding can also occur with an antisense sequence of the internal loop of 5BSL3.2, supporting previous findings that suggested that the sequence centered on nucleotide 9110 was likely unstructured (Diviney et al 2008). Together, these results support a view in which recognition between the internal loop of 5BSL3.2 and the sequence centered on nucleotide 9110 would not require structured motifs to occur, in contrast to what has been proposed recently (Shetty et al 2013). The results raise the problem of the kinetics of formation of these complexes.…”

“…1B). The SL2 region is, therefore, involved in two different interactions that are likely exclusive, leading consequently to different activities, translation, replication, or encapsidation during the viral replication, as also suggested by a work published recently during the review of our manuscript (Shetty et al 2013).…”

Dissecting noncoding and pathogen RNA–protein interactomes

“…3D), compatible with the RNAbinding properties of PCBP2 (Du et al 2005). Based upon its noncoding binding topology, PCBP2 appears to have broad regulatory scope through interaction with 18S rRNA as well as serving as a translational regulator in both cancer (Eiring et al 2010;Han et al 2013) and infectious disease (Gamarnik and Andino 2000;Herold and Andino 2001;Shetty et al 2013).…”

“…We applied this Fully Automated and Standardized iCLIP (FAST-iCLIP) pipeline to a published iCLIP data set of the RBP hnRNP-C, recapitulating the published biology of this protein. We then applied FAST-iCLIP to the human and viral interactomes of Poly-C binding protein 2 (PCBP2), a KH domain (hnRNP-K homology) (Lunde et al 2007) containing RBP that has a preference for C/Urich motifs (Choi et al 2009) as well as pathological associations in cancer (Eiring et al 2010;Han et al 2013) and infectious disease (Gamarnik and Andino 2000;Herold and Andino 2001;Shetty et al 2013). The unbiased characterization of the PCBP2 protein revealed novel RNAprotein interactions and provided supporting evidence for previously known cellular and viral roles for PCBP2.…”