Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements

Abstract

The translation of picornavirus genomic RNAs occurs by a cap-independent mechanism that requires the formation of specific ribonucleoprotein complexes involving host cell factors and highly structured regions of picornavirus 59 noncoding regions known as internal ribosome entry sites (IRES). Although a number of cellular proteins have been shown to be involved in picornavirus RNA translation, the precise role of these factors in picornavirus internal ribosome entry is not understood. In this report, we provide evidence for the existence of distinct mechanisms for the internal initiation of translation between type I and type II picornavirus IRES elements. In vitro translation reactions were conducted in HeLa cell cytoplasmic translation extracts that were depleted of the cellular protein, poly(rC) binding protein 2 (PCBP2). Upon depletion of PCBP2, these extracts possessed a significantly diminished capacity to translate reporter RNAs containing the type I IRES elements of poliovirus, coxsackievirus, or human rhinovirus linked to luciferase; however, the addition of recombinant PCBP2 could reconstitute translation. Furthermore, RNA electrophoretic mobility-shift analysis demonstrated specific interactions between PCBP2 and both type I and type II picornavirus IRES elements; however, the translation of reporter RNAs containing the type II IRES elements of encephalomyocarditis virus and foot-and-mouth disease virus was not PCBP2 dependent. These data demonstrate that PCBP2 is essential for the internal initiation of translation on picornavirus type I IRES elements but is dispensable for translation directed by the structurally distinct type II elements.

Cytoplasmic Relocalization of Heterogeneous Nuclear Ribonucleoprotein A1 Controls Translation Initiation of Specific mRNAs

“…Here, we have shown that rhinovirus infection exerts a direct positive switch to control its IRES-mediated translation through relocalization of an ITAF. Several other ITAFs have been described for the rhinovirus IRES, including unr, hnRNP I/PTB, and hnRNP E2 (Hunt et al, 1999;Walter et al, 1999;Boussadia et al, 2003). However, the in vivo role of these ITAFs in HRV IRES function after rhinovirus infection, as well as their respective role in IRES function in general, may warrant further investigations.…”

Picornavirus IRES elements: RNA structure and host protein interactions

“…In particular, PCBP2 stimulates the activity of PV, HRV and CBV3 IRES (Gamarnik et al, 2000;Sean et al, 2009). However, although PCBP2 binds to both EMCV and FMDV IRES, this factor only stimulates the first one (Walter et al, 1999).…”

The poly(C)-binding proteins: A multiplicity of functions and a search for mechanisms

“…The role of aCP-2 in translational enhancement may be shared by other picornaviruses+ Cap-independent translation initiation within the picornavirus family is mediated by two major classes of IRESs (type I and type II)+ These IRESs differ in their sequences, secondary structures, and biological properties (Wimmer et al+, 1993;Ehrenfeld, 1996)+ The enteroviruses and rhinoviruses utilize type I IRESs, and the cardioviruses and aphthoviruses utilize the type II IRES elements+ Although aCP-2 is capable of interacting in vitro with both type I IRESs (from coxsackievirus strain B and human rhinovirus) and type II IRES (from encephalomyocarditis virus and foot-and-mouth disease virus), an effect on translation of the cardiovirus and aphthovirus has not been demonstrated+ These data suggest that a functional role for aCP-2 in binding to viral 59 UTRs appears to be limited to type I IRES elements (Walter et al+, 1999)+ aCP plays a role in the translation of viruses in addition to those in the picornavirus family+ Although the IRES element of hepatitis A virus (HAV) cannot easily be classified as a type I or type II IRES (Brown et al+, 1994), aCP-2 is required to facilitate HAV internal ribosome entry similarly to type I IRESs (Graff et al+, 1998)+ Along the same lines, the hepatitis C virus (HCV) IRES has been difficult to classify as a type I or type II IRES due to its unique structure (Reynolds et al+, 1995)+ Whereas aCP-1 and aCP-2 can bind specifically to the HCV 59 UTR (Spangberg & Schwartz, 1999), it has not been possible to document an effect of this binding on HCV translation (Fukushi et al+, 2001)+ In this respect, the HAV IRES behaves in a manner reminiscent of a type I IRES, whereas HCV IRES resembles a type II IRES+ Thus, aCP binding can contribute to both silencing and enhancement of translation+ The former action is mediated by 39 UTR complexes and the latter by complexes within the 59 IRES segments of picornaviruses+ In neither case are the downstream events that control the rates of ribosome loading clearly defined+ In both cases, the complexes are formed at substantial distances from the sites of translation initiation+ Whether the two processes converge on a common pathway remains an interesting possibility+ Delineation of the 274…”