RNA Determinants of Picornavirus Cap-Independent Translation Initiation

Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo

“…In agreement with the observation that internal initiation of translation depends upon IRES binding to cellular trans-acting factors, RNA-protein interactions have been described to occur in several IRES elements (reviewed in Stewart & Semler, 1997;Andino et al+, 1999)+ Presently, the biological role that some of these transacting factors play in the internal recruitment of the translational machinery is under intense study+ The IRES of pestivirus and hepatitis C (HCV) binds eIF3 directly (Buratti et al+, 1998;Pestova et al+, 1998;Sizova et al+, 1998), an observation that has led to the assumption that these IRES elements use a prokaryotic-like mode of interaction between the mRNA and the ribosome+ By contrast, the translation initiation factors eIF4A, eIF4B, eIF4G, eIF3, and eIF2, but not eIF4E, are necessary to mediate encephalomyocarditis (EMCV) IRES-dependent complex initiation in a reconstituted system with purified translation initiation factors (Pestova et al+, 1996a; reviewed in Pestova & Hellen, 1999)+ Identification of the eIF4G-binding site in the EMCV IRES by chemical and enzymatic footprinting (Kolupaeva et al+, 1998) was consistent with the observation that the initiation factor eIF4A and the central domain of eIF4G are sufficient to mediate internal entry of 43S preinitiation complexes (Pestova et al+, 1996b)+ However, up to now, the binding site of eIF4G in other IRES elements is not known, and more importantly, it remains to be elucidated whether eIF4G-RNA interaction plays a biological role on IRES-dependent translation initiation in vivo+…”

A tertiary structure model of the internal ribosome entry site (IRES) for methionine-independent initiation of translation

“…Internal ribosome entry is a mode of translation initiation+ It is thought that an internal sequence element of mRNA recruits ribosomes onto an upstream region of the coding sequence, and therefore translation occurs independently of the 59 cap+ The RNA element that is responsible for ribosome entry is called the internal ribosome entry site (IRES)+ The first IRES elements were found in picornaviruses (Jang et al+, 1988;Pelletier & Sonenberg, 1988)+ Then others were found in several viral and cellular mRNAs, such as hepatitis C virus (Tsukiyama-Kohara et al+, 1992), cowpea mosaic virus (Thomas et al+, 1991), immunoglobulin heavy chain-binding protein (Macejak & Sarnow, 1991), and fibroblast growth factor 2 (Vagner et al+, 1995)+ For the IRES elements in cellular mRNA, a short nucleotide segment complementary to the 18S rRNA sequence has functional importance (Chappell et al+, 2000)+ In the viral IRES elements, the secondary and tertiary structure in the IRES has critical roles for translation initiation+ Three types of viral IRES elements are well characterized: those of the entero/rhinovirus, cardio/ aphthovirus, and hepatitis C virus groups+ These characterized IRES elements contain several stem-loop structures, and there are possible tertiary structure interactions between loops and bulges+ Within each group, the secondary structure is conserved, but there is almost no conservation between the groups (reviewed in Jackson & Kaminski, 1995;Lemon & Honda, 1997;Stewart & Semler, 1997)+ Cricket paralysis-like viruses (CrPV-like viruses) are a group of positive-stranded RNA viruses that infect insects+ These viruses are morphologically similar to picornaviruses; however, recently reported genome sequences have revealed that CrPV-like viruses have two open reading frames (ORFs), unlike picornaviruses+ To date, the genome sequences of seven CrPV-like viruses have been reported: Drosophila C virus (DCV; Johnson & Christian, 1998), Rhopalosiphum padi virus (RhPV; Moon et al+, 1998), Plautia stali intestine virus (PSIV; Sasaki et al+, 1998), himetobi P virus (HiPV; Nakashima et al+, 1999), Triatoma virus (TrV; Czibener et al+, 2000), cricket paralysis virus (CrPV; Wilson et al+, 2000a), and black queen-cell virus (BQCV; Leat et al+, 2000)+ The first ORF encodes a nonstructural protein precursor and the precursor is produced by IRESmediated translation (Wilson et al+, 2000a)+ The second ORF encodes a capsid protein precursor, and this precursor is also produced by IRES-mediated translation Domier et al+, 2000;…”

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

“…Translation initiation of a majority of cellular and viral mRNAs begins with the recognition of the m 7 GTP cap at the 59 terminus of the mRNA by cellular factors that associate with the 43S preinitiation complex to recruit the small ribosomal subunit to the mRNA+ Following 59 cap recognition, the 43S preinitiation complex scans the 59 noncoding region (59 NCR) of the mRNA for an AUG codon that is in the appropriate context for the recruitment of the large ribosomal subunit and subsequent initiation of translation (Kozak, 1987(Kozak, , 1989)+ Thus, RNAs possessing long, highly structured 59 NCRs encoding multiple AUG codons in favorable contexts upstream of the initiator AUG or RNAs lacking m 7 GTP cap structures should not be capable of efficient initiation of translation by a cap-dependent, ribosome-scanning mechanism+ RNAs containing such characteristics belong to an expanding group of viral and cellular RNAs capable of initiating translation by alternative mechanisms+ These mRNAs can be divided into two subsets based on the mechanism by which translation is initiated+ The first subset is characterized by the capdependent recruitment of ribosomes followed by nonlinear migration of these ribosomes ("ribosome shunting") to the initiator AUG of the mRNA+ Cauliflower mosaic virus and adenovirus RNAs have been shown to utilize ribosome shunting for the initiation of translation (Futterer et al+, 1993;Yueh & Schneider, 1996)+ The second subset of alternatively translated RNAs is comprised of RNAs translated by a mechanism of cap-independent, direct internal ribosome bind-ing and entry+ This group contains many cellular and viral RNAs including the genomic RNAs of a family of animal viruses, the Picornaviridae (Jang et al+, 1988;Pelletier & Sonenberg, 1988; for reviews, see Jackson & Kaminski, 1995;Stewart & Semler, 1997)+…”

“…In fact, much of the evidence compiled to date suggests that the internal initiation of translation on both type I and type II picornavirus IRES elements requires certain trans-acting cellular proteins in addition to canonical translation initiation factors (for reviews, see Jackson & Kaminski, 1995;Stewart & Semler, 1997)+ In this study, we investigated the process of translation as directed by the IRES elements of poliovirus (PV), coxsackievirus strain B (CVB), human rhinovirus (HRV), encephalomyocarditis virus (EMCV), and foot-andmouth disease virus (FMDV)+ Specifically, we examined whether these picornaviruses, representing four of the six picornavirus genera, require the cellular protein, PCBP2, for viral translation+ PCBP2 is a cellular RNA-binding protein that interacts with the 59 NCR of poliovirus RNA (Dildine & Semler, 1992;Blyn et al+, 1995Blyn et al+, , 1996+ The protein contains three hnRNP K-homologous (KH) RNA-binding domains and has a binding preference for poly(rC)+ PCBP2 mRNA is widely expressed and has been localized both in the cytoplasm and the nucleus (Leffers et al+, 1995)+ To date, the only known functions attributed to PCBP2 are its functional associations with the 39 NCRs of the a-globin and the tyrosine hydroxylase mRNAs in complexes that impart stability (Kiledjian et al+, 1995;Paulding & Czyzyk-Krzeska, 1999)+ Similar complexes have also been found to form on the 39 NCRs of lipoxygenase, a(I)-collagen, and erythropoietin mRNAs (Holcik & Liebhaber, 1997;Czyzyk-Krzeska & Bendixen, 1999)+ In addition to the role of PCBP2 in cellular mRNA stability, a number of additional functions relating to viral infection have been ascribed to the protein+ We have previously shown that the interaction of PCBP2 with the poliovirus 59 NCR performs dual functions in the life cycle of the virus by facilitating the initiation of both viral protein synthesis and viral RNA synthesis (Blyn et al+, 1997;Parsley et al+, 1997)+ Likewise, Andino and colleagues confirmed this functional duality and proposed that PCBP could also function in a mechanism involving accessory proteins of viral origin to regulate the processes of translation and replication (Gamarnik & Andino, 1997)+ In addition to its documented functions in the poliovirus life cycle, PCBP2 also plays a role in the translation of HAV genomic RNA (Graff et al+, 1998)+ Furthermore, human papillomavirus type 16 (HPV16), a member of the Papovaviridae, has evolved a mechanism to utilize PCBP2+ For HPV16, PCBP2 acts as a regulator of late gene expression (Collier et al+, 1998)+ This work investigates the possibility that RNA binding by PCBP2 is a general requirement for the internal initiation of translation on picornavirus IRES elements+ Competition RNA mobility-shift assays conducted with 32 P-labeled PV stem-loop IV RNA, recombinant PCBP2 (rPCBP2), and five different picornavirus 59 NCRs confirmed a conserved physical interacti...…”