Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation

Abstract

Poliovirus (PV) is a medically important member of the picornavirus genus Enterovirus, which is collectively responsible for millions of human infections per year worldwide. Enteroviruses contain a single-stranded RNA genome that is a direct template for translation by the host cell's translational machinery, and this translation step is required for host morbidity and progeny virus production. The ϳ7.5-kb PV genome is comprised of a single open reading frame encoding a 247-kDa polyprotein which is processed to 11 final products, a short 3Ј untranslated region (UTR) with defined RNA structure, a poly(A) tail of strain-specific length that is approximately 80 to 120 nucleotides long, and a ϳ700-nucleotide 5Ј UTR containing an internal ribosome entry site (IRES) for facilitation of viral translation. The extreme 5Ј end of the PV genome, known as the cloverleaf or oriL, has roles in nucleic acid stability (5), translation (54), and viral genomic RNA replication (43,59). Notably, the viral genome has no 7-methylguanosine cap.The viral IRES is responsible for driving translation, and the 7-methylguanosine cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is not required for IRES-dependent translation. The large scaffolding protein eIF4G is cleaved early in PV infection, and the fragments produced cannot support capdependent translation (40); however, cleaved eIF4G stimulates IRES-driven translation more than intact eIF4G (12,25,41). The PV IRES is thought to bind eIF4G directly, as it does to related viral IRESs such as that of encephalomyocarditis virus (34). The C-terminal fragment of eIF4G that functions in IRES-dependent translation (p100) is able to recruit eIF3 and the 40S ribosomal subunit to the viral RNA. Other canonical translation factors involved in PV IRES-mediated translation include the DEAD box helicase eIF4A and its activator eIF4B, which also directly binds the IRES (47). The poly(A) tail/ poly(A)-binding protein (PABP) interaction with eIF4G is also proposed to stimulate PV IRES translation by mediating a closed-loop structure and enhanced interactions between PABP and eIF4GI (7,13,45,57).In addition to these canonical translation factors, other cellular proteins, IRES trans-activating factors (ITAFs), stimulate IRES translation. These include polypyrimidine tract-binding protein (PTB) (26, 50), poly r(C)-binding protein 2 (PCBP2) (9, 10), lupus autoantigen (La) (44), and upstream of N-ras (Unr) (16). The ITAFs map to multiple binding sites in enteroviral IRES structures (9,18,23) and are proposed to have roles as chaperones to hold the IRES in a translation-competent conformation and possibly to also act as de facto signals for the switch from translation to replication. Additionally, ITAFs may recruit effector proteins to the IRES, as seen in the recent discovery that SRp20 binds PCBP2 and is required for IRES-mediated translation (6).The two viral proteinases, 2A proteinase (2A pro ) and 3C proteinase (3C pro ), cleave several cellular proteins during infection in addition to viral p...

Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication

“…Although not shown as a cleaved polypeptide in Fig. 7, host cell poly(A) binding protein (PABP) has also been shown to be cleaved by poliovirus 3C or 3CD proteinase, contributing to the shutdown of cap-independent viral translation (37). With the increased concentration of viral proteins, RNA replication complexes are formed, leading to cytoplasmic foci with higher concentrations of cleaved PCBP2 than of full-length PCBP2.…”

Poliovirus Switches to an eIF2-Independent Mode of Translation during Infection

“…Cells were then lysed in passive lysis buffer or TRIzol reagent (Invitrogen) for analysis of firefly luciferase or reporter mRNA, respectively, by reverse transcription (RT)-PCR to standardize the results. Reporters for electroporation were transcribed in vitro using T7 RNA polymerase (Invitrogen) according to the manufacturer's protocols from ApaI-linearized pT7-FLuc, HindIII-linearized pT7-PV 5Ј-FLuc-3Ј A 71 (9) and NaeI-linearized pCMV-RLuc-IGR-FLuc (CMV stands for cytomegalovirus). HeLa S3 and 293T cells were electroporated using the Neon electroporation system (Invitrogen) according to the manufacturer's protocols prior to treatment with sodium arsenite (Sigma) or Sal003 (kind gift of Mauro Costa-Mattioli) at the indicated concentrations.…”

Picornavirus IRES elements: RNA structure and host protein interactions

“…The mechanisms by which picornavirus infections achieve inhibition of cellular mRNAs translation reside mainly in the cleavage of host translation initiation factors (eIFs) by proteases L, 2A or 3C, as well as in the phosphorylation of eIF2␣ and other translation factors (Walsh and Mohr, 2011). Specifically, cleavage of eIF4GI, eIF4GII, eIF4AI, eIF3a, eIF5B, and poly(A) binding protein (PABP) (Belsham et al, 2000;Bonderoff et al, 2008;de Breyne et al, 2008a;Gradi et al, 2004;Lamphear et al, 1993;Li et al, 2001;Rodriguez Pulido et al, 2007) induces the inhibition of host protein synthesis and, in general, a shut-down of cellular gene expression.…”