The N-terminal K Homology Domain of the Poly(rC)-binding Protein Is a Major Determinant for Binding to the Poliovirus 5′-Untranslated Region and Acts as an Inhibitor of Viral Translation

Abstract

The poly(rC)-binding proteins (PCBP1 and PCBP2) are RNA-binding proteins whose RNA recognition motifs are composed of three K homology (KH) domains. These proteins are involved in both the stabilization and translational regulation of several cellular and viral RNAs. PCBP1 and PCBP2 specifically interact with both the 5-element known as the cloverleaf structure and the large stem-loop IV RNA of the poliovirus 5-untranslated region. We have found that the first KH domain of PCBP2 (KH1) specifically interacts with the viral RNAs, and together with viral protein 3CD, KH1 forms a high affinity ternary ribonucleoprotein complex with the cloverleaf RNA, resembling the full-length PCBP protein. Furthermore, KH1 acts as a dominant-negative mutant to inhibit translation from a poliovirus reporter gene in both Xenopus laevis oocytes and HeLa cell in vitro translation extracts.Translation in eukaryotic cells is a highly regulated process involving a complex protein machinery. There is increasing evidence that translation of several mRNAs is determined by the specific and regulated interaction of certain proteins with RNA elements in the 3Ј-and 5Ј-untranslated regions (for reviews, see Refs. 1 and 2). Although many of these cis-acting RNA elements have been defined, only a few trans-acting regulatory proteins are known, and the mechanisms by which they regulate translation are poorly understood.As with cellular messages, translation of viral RNA is also subjected to complex regulation. For example, for positive strand RNA viruses, the genomic RNA is utilized as a template for translation, RNA replication, and formation of new virions; hence, the usage of the RNA must be regulated. For poliovirus, this regulation seems to be dependent on signals within the 5Ј-UTR.1 Whereas the majority of cellular mRNAs depend on the 5Ј-cap structure to initiate translation, poliovirus initiates translation internally via a cap-independent mechanism from an RNA element termed the internal ribosomal entry site (IRES) located within the 5Ј-UTR. Computer modeling and biochemical analysis suggest that the secondary structure of the poliovirus 5Ј-UTR is composed of six distinct domains, stem-loops I-VI (see Fig. 1A). In addition to the canonical initiation factors, poliovirus translation initiation requires additional host cell factors, some of which appear to interact directly with the viral RNA (reviewed in Refs. 3 and 4). Among these essential factors are PCBP1 and PCBP2, two closely related poly(rC)-binding proteins, (also referred to as hnRNPs E1 and 2 or ␣CP1 and ␣CP2). These proteins facilitate viral translation through the interaction with both the first stemloop domain (which folds into a cloverleaf-like structure) and stem-loop IV of the poliovirus 5Ј-UTR (5-8). Both PCBP1 and PCBP2 form a low affinity complex with the cloverleaf RNA, but together with viral protein 3CD (the precursor of the viral polymerase 3D and the viral protease 3C), they are incorporated into a high affinity ternary ribonucleoprotein complex (9, 10). Ternary compl...

Prenylome profiling reveals S-farnesylation is crucial for membrane targeting and antiviral activity of ZAP long-isoform

“…S3 A and B) and consistent with previous reports of farnesylation on this chaperone protein (20). In addition, we demonstrate that HA-tagged Pcbp1, a protein implicated in nucleic acid binding (41), is also labeled by alk-FOH, the majority of which appears to be on Cys355 at the carboxyl terminus and insensitive to prenylation inhibitors (Fig. 3 C and D).…”

Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES

“…3). This confirms previous studies showing that the KH1 domain is required for poliovirus stem-loop IV RNA binding (Silvera et al 1999;Walter et al 2002). If ⌬KH1-PCBP2 interacts with wild-type PCBP2, but does not bind to stem-loop IV RNA, we hypothesized that ⌬KH1-PCBP2 would act as a dominant negative in poliovirus translation.…”

“…Full-length PCBP2 is required for optimal RNA binding and translation; however, the KH1 domain is necessary and sufficient for binding to stem-loop I and stem-loop IV of poliovirus RNA (at ∼10 µM recombinant protein) and can act as a dominant negative for poliovirus translation in Xenopus oocytes (Silvera et al 1999). The KH3 domain increases RNA binding efficiency and is important for poliovirus translation, but not RNA replication (Walter et al 2002).…”

Optimized RNA Targets of Two Closely Related Triple KH Domain Proteins, Heterogeneous Nuclear Ribonucleoprotein K and αCP-2KL, Suggest Distinct Modes of RNA Recognition

“…Remarkably, ␣CP can also mediate translational enhancement. In this case ␣CP increases the efficiency of cap-independent translation of picornavirus RNA by binding to two specific sites within the 5Ј-UTR internal ribosome entry site as follows: the 5Ј-terminal cloverleaf structure of the 5Ј-UTR (38,39) and stem-loop IV (38,40). The interaction with the 5Ј cloverleaf, which is dependent on the co-binding of the viral protein 3CD, also controls the switch from translation to replication of the polio RNA viral genome (41).…”