Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.

Abstract

The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for capindependent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.

MgATP binding to the nucleotide‐binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate‐binding domains

“…Precursor mass spectra and CID product ion spectra were collected as described previously (Blyn et al, 1996;Stahl et al, 1996). Product ion spectra were correlated with all entries of the OWL nonredundant protein database version 26.3 using the SEQUEST database correlation program version B22.…”

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

“…PCBP2 was first identified as a 38-kDa cellular protein that interacts with the stem-loop IV domain within the 5Ј noncoding region of poliovirus RNA and is required for poliovirus translation (Blyn et al 1996. Depletion of PCBP2 from HeLa cytoplasmic extracts severely decreased poliovirus translation, and the addition of recombinant PCBP2 to depleted extracts restored translation to mock depleted levels, confirming that PCBP2 is necessary for efficient poliovirus translation.…”

“…PCBP2 has also been implicated in poliovirus RNA replication. Andino and colleagues first reported a cellular protein that bound to the poliovirus RNA cloverleaf structure (stem-loop I) in conjunction with the viral protein 3CD and was necessary for RNA replication (Andino et al 1990(Andino et al , 1993Blyn et al 1996). This cellular protein was later identified as PCBP2 and was confirmed to have a role in poliovirus RNA replication (Gamarnik and Andino 1997;Parsley et al 1997;Walter et al 2002).…”

“…Examples of these proteins include polypyrimidine tract binding protein (PTB), La autoantigen, and upstream of n-ras protein (unr; for review, see Jackson 2002). One cellular protein, poly(rC) binding protein 2 (PCBP2), has been shown to bind to the 5Ј noncoding region of poliovirus RNA and to be necessary for the translation of picornaviruses containing type I IRES elements (Blyn et al 1996Gamarnik and Andino 1997;Walter et al 1999).…”

Genetic Analysis of a Poliovirus/Hepatitis C Virus (HCV) Chimera: Interaction between the Poliovirus Cloverleaf and a Sequence in the HCV 5′ Nontranslated Region Results in a Replication Phenotype

“…3A) serves as a cis-acting signal in PV RNA replication (reviewed in reference 27). It forms an RNP of unknown structure with the PV proteinase 3CD pro (2,3) and either PV polypeptide 3AB (10,26,27) or cellular RNA binding poly(rC) binding protein (4,7,18). Genetic analysis has identified stem-loop D as the binding domain of 3CD pro (3; E. Rieder, W. Xiang, and E. Wimmer, unpublished data).…”