Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates

Picornaviruses are a group of small, positive-sense RNA viruses that replicate in the host cell cytoplasm. Their genomes are characterized by highly structured 5′ noncoding regions (NCRs) containing a cruciform RNA structure (termed stem-loop I, or cloverleaf) necessary for viral-RNA replication and an internal ribosome entry site (IRES) that allows translation of a virus polyprotein from the single open reading frame of genomic RNA. The virus polyprotein is cleaved by encoded viral proteinases (30) to yield the mature structural and nonstructural proteins, including the RNA-dependent RNA polymerase 3D. Due to the limited size of the genome, picornaviruses have evolved to utilize host cell factors in concert with their own virus-encoded proteins and RNA secondary structures to efficiently drive the replication cycle. Poliovirus, an example of the viruses using such a combination of host and viral functions, is the causative agent of paralytic poliomyelitis and the most extensively studied picornavirus. Cellular proteins play an important role in the replication of the poliovirus RNA genome (19, 37). For example, poly(rC) binding protein 2 (PCBP2) binds to the stem-loop I RNA cruciform structure at the 5′ end of the genome. Binding of PCBP2 to this region is required for viral-RNA replication, and disruption of the capability of the protein to bind to stem-loop I disrupts RNA replication (55). During the synthesis of poliovirus negative-strand RNA intermediates, it has been proposed that the 5′ and 3′ ends of poliovirus positive-sense RNA communicate via interactions formed by host cell protein poly(A)-binding protein (PABP) bound to the poly(A) tract at the 3′ end of the genome and PCBP2 and the virus-encoded 3CD protein bound to the stem-loop I RNA structure (5, 20, 28). The 3′ NCR of poliovirus genomic RNA is located immediately following the 3D polymerase coding region and is relatively short (71 nucleotides) compared to the 5′ NCR (742 nucleotides). For poliovirus, the 3′ NCR consists of two stem-loop structures (termed X and Y) that are formed by base-pairing interactions between the nucleotides of the 3′ terminus and the genetically encoded poly(A) tail. These stem-loop structures have been predicted to participate in tertiary “kissing” interactions between the RNA stem-loops themselves (31, 40) and/or to bind cellular factors (48, 53) to facilitate RNA synthesis. Previous studies by Brown et al. characterized a viable poliovirus mutant lacking the 3′ NCR (PVΔ3′NCR) that exhibited a growth defect in cultured human cell lines (12, 13). Due to the proximity of the 3′ NCR to the presumed site of negative-strand RNA synthesis initiation at the 3′ poly(A) tail, it was originally thought that the observed growth defect was due to a deficiency in negative-strand RNA synthesis owing to a lack of 3′ NCR stem-loop structures. However, further examination of the strand-specific synthesis during PVΔ3′NCR infection of HeLa cells revealed that while negative-strand RNA synthesis was largely unchanged compared to the wild type, positive-strand RNA synthesis was diminished significantly. This suggested either that a terminal RNA structure (e.g., the 3′ NCR) can act in trans to influence the initiation of RNA synthesis at the opposite end of an RNA strand of negative polarity or that the negative-strand complement of the 3′ NCR at the 5′ end of the replicative intermediate acts in cis to mediate positive-strand RNA synthesis. In addition, the observation that the PVΔ3′NCR positive-strand RNA synthesis defect was further exacerbated in a neuroblastoma cell line compared to HeLa cells indicated that a limiting cellular factor interacting with the 3′ NCR might play a role in RNA replication. Previous studies had identified the interaction of the nuclear protein nucleolin with the 3′ NCR (51, 53). It has also been reported that recombinant encephalomyocarditis virus (EMCV) 3Dpol, as well as the poliovirus polymerase precursor 3CD, bind this region of the genome (18, 26). As there has yet to be a function ascribed to the binding of these proteins to the poliovirus 3′ NCR, we hypothesized that the 5′ end of negative-strand RNA (the complement to the 3′ NCR) interacts with additional viral and/or cellular factors to promote efficient positive-strand RNA synthesis. The 5′ end of poliovirus negative-strand RNA has been predicted by RNA-folding algorithms (59) to adopt stem-loop structures highly similar to those of its positive-strand equivalent (Fig. ​(Fig.1A),1A), including base pairing with the poly(U) tract in negative-strand RNA. Initial RNA-protein binding assays carried out in our study using radiolabeled RNA probes corresponding to the 5′ end of negative-strand RNA, here denoted 5′(−), identified distinct ribonucleoprotein (RNP) complexes in extracts from poliovirus-infected HeLa cells. The appearance and pattern of these complexes were similar to those observed in previous studies using the opposite terminus of the poliovirus replicative intermediate, the 3′ end of negative-strand RNA [3′(−)]. Roehl and Semler first reported the appearance of these complexes using UV cross-linking approaches, and the identity of the 36-kDa protein component of such complexes was later determined by mass spectrometry to be the cellular protein heterogeneous nuclear ribonucleoprotein C (hnRNP C) by Brunner et al. (15, 43). hnRNP C is a predominantly nuclear protein that is highly abundant in human cells and exists as two alternatively spliced isoforms, C1 and C2 (22) (Fig. ​(Fig.1B).1B). In the cell, hnRNP C forms a heterotetramer consisting of three C1 molecules and a single C2 molecule. This arrangement is thought to orient the amino-terminal high-affinity RNA recognition motifs (RRM) to bind RNA. hnRNP C functions in cellular mRNA biogenesis processes in the nucleus, including mRNA splicing and stabilization of pre-mRNA (22, 46). FIG. 1. The 5′ end of poliovirus negative-strand RNA and hnRNP C proteins. (A) Mfold-predicted structure of the 5′ end of poliovirus negative-strand RNA, the complement of the genomic-strand 3′ NCR. Shown are the X(−) stem-loop ... In this study, we used RNA binding assays in conjunction with the immunoprecipitation (IP) of RNP complexes by hnRNP C antibodies to demonstrate that hnRNP C binds to the 5′ end of poliovirus negative-strand RNA, providing evidence that hnRNP C can bind both termini of viral negative-strand RNA intermediates. We further show that addition of recombinant wild-type hnRNP C1 protein, but not C1 proteins harboring a mutation in the multimerization domain (56), to poliovirus in vitro translation/replication assays can stimulate poliovirus RNA synthesis, suggesting that hnRNP C binding as a multimer may be important for its participation in RNA replication during virus infection. In addition, we used retrovirus-mediated expression of hnRNP C-specific short hairpin RNAs (shRNAs) to significantly reduce the levels of hnRNP C proteins in HeLa cells. Poliovirus yields were decreased 5-fold in hnRNP C-specific shRNA-treated cells compared to infections of control shRNA-treated cells. Using quantitative real-time PCR analysis, we determined that the accumulation of positive-strand RNA is selectively decreased in hnRNP C-depleted cells during the early phases of poliovirus infection, implying a role for hnRNP C proteins in virus positive-strand RNA synthesis. Our results suggest that cellular hnRNP C proteins play an important role in the formation of positive-strand RNA replication complexes in infected cells.