The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing sitedirected lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.Following entry into a host cell, the single-stranded, positivesense genomic RNA of poliovirus (PV) must provide a nucleation point for the formation of numerous ribonucleoprotein (RNP) complexes. These complexes are capable of mediating the three major processes that the RNA must undergo to establish a successful infection of the host cell. The PV genomic RNA must be translated, replicated, and packaged for a productive virus infection to ensue. Given the small size (ϳ7,400 nucleotides [nt]) of the PV genome, the virus has evolved to utilize numerous proteins or other molecular machinery resident in the host cell to carry out its infectious cycle. One example of the molecular scavenging necessitated by the limited coding capacity of PV is utilization of a single cellular RNA-binding protein, poly(rC) binding protein 2 (PCBP2), for the formation of RNP complexes that form on two different PV 5Ј noncoding region (5Ј NCR) RNA secondary structures to mediate two of the three above-mentioned processes (16,24,25,50).PCBP2 (also known as hnRNP E2 and ␣CP-2) is a cellular RNA-binding protein that interacts with the 5Ј NCR of PV RNA (14,15,23). The protein contains three hnRNP K-homology (KH) RNA-binding domains and has a binding preference for poly(rC). In in vitro binding studies, the first and third KH domains ap...

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To study the role of the RNA polymerase domain (3D) in the proteinase substrate recognition and RNA binding properties of poliovirus polypeptide 3CD, we generated recombinant 3C and 3CD polypeptides and purified them to near homogeneity. By using these purified proteins in in vitro cleavage assays with structural and non-structural viral polyprotein substrates, we found that 3CD processes the poliovirus structural polyprotein precursor (P1) 100 to 1000 times more efficiently than 3C processes P1. We also found that trans-cleavage of other 3CD molecules and sites within the non-structural P3 precursor is more efficiently mediated by 3CD than 3C. However, 3C and 3CD appear to be equally efficient in the processing of a non-structural polyprotein precursor, 2C3AB. Four mutated 3CD polyproteins with site-directed lesions in the 3D domain of the proteinase were analyzed for their ability to process viral polyprotein precursors and to form a ternary complex with RNA sequences encoded in the 5 terminus of the viral genome. Analysis of mutated 3CD polypeptides revealed that specific mutations within the 3D amino acid sequences of 3CD confer differential effects on 3CD activity. All four mutated 3CD proteins tested were able to process the P1 structural precursor with wild type or near wild type efficiency. However, three of the mutated enzymes demonstrated an impaired ability to process some sites within the P3 non-structural precursor, relative to wild type 3CD. One of the mutant 3CD polypeptides, 3CD-3DK127A, also displayed a defect in its ability to form a ternary ribonucleoprotein complex with poliovirus 5 RNA sequences.The positive strand RNA genome of poliovirus contains a single open reading frame that codes for a 247-kDa viral polyprotein. Proper expression of poliovirus gene products requires specific protein processing of the viral polyprotein by the enzymatic activities of virally encoded proteinases. These enzymes cleave the polyprotein co-and post-translationally into the structural and non-structural viral gene products (Fig. 1). The majority of cleavage events within the viral polyprotein occur at Q-G bonds and is mediated by a proteinase function associated with the viral-encoded 3C protein (1-6). The rate at which 3C pro1 -mediated proteolytic processing occurs at various sites within the viral polyprotein controls the temporal expression of viral gene products (7). This regulation of gene expression is essential for viral replication since polyprotein precursors have functions in replication that are distinct from those of the mature cleavage products (8, 9). An example of a molecule exhibiting these differential functions is the viral polyprotein 3CD, which is an active proteinase containing the entire amino acid sequences of the 3C proteinase and the RNA-dependent RNA polymerase, 3D. Polypeptide 3CD is a multi-functional protein required for both proteolytic processing of the capsid precursor protein and, although it has no detectable elongation activity in vitro, viral RNA replication. The role of 3...

Hypoviruses persistently alter multiple phenotypic traits, stably modify gene expression, and attenuate virulence (hypovirulence) of their pathogenic fungal host, the chestnut blight fungus Cryphonectria parasitica. The pleiotropic nature of these changes is consistent with hypovirus-mediated perturbation of one or more cellular signal transduction pathways. We now report that two hypoviruses that differ in the severity of symptom expression differentially perturb specific cellular signaling pathways. The C. parasitica 13-1 gene, originally identified as a hypovirus-inducible and cyclic AMP (cAMP)-regulated gene, was used to design a promoter-GFP reporter construct with which to monitor perturbation of cAMP-mediated signaling. Virusmediated modulation of calcium/calmodulin/inositol trisphosphate-dependent signaling was monitored by measuring transcript accumulation from the C. parasitica laccase gene, lac-1. Infection by the severe hypovirus strain CHV1-EP713 caused a substantial induction of 13-1 promoter activity and a reduction of total extracellular laccase enzymatic activity (LAC-1 and LAC-3). In contrast, 13-1 promoter activity and total laccase activity were only marginally altered upon infection with the mild hypovirus strain CHV1-Euro7. However, examination of lac-1-specific transcript accumulation under previously defined culture conditions revealed that both CHV1-EP713 and CHV1-Euro7 perturbed calcium/calmodulin/inositol trisphosphate-dependent signaling. CHV1-EP713/CHV1-Euro7 chimeric viruses were used to map viral determinants responsible for modulation of cAMP-dependent signaling to domains within the central portion of the second open reading frame.Hypoviruses were initially discovered and have been extensively studied because they reduce the virulence (hypovirulence) of their fungal host, the chestnut blight fungus Cryphonectria parasitica (1,35,38,39,40,51). However, it was observed early on in the characterization of hypovirulent C. parasitica field strains that phenotypic changes associated with hypovirus infection were not confined to virulence attenuation. Although different hypoviruses cause different constellations of phenotypic changes, the symptoms caused by a specific hypovirus are stable and generally consistent in different C. parasitica strain genetic backgrounds (1,2,4,21,22). For example, phenotypic changes associated with hypovirulence caused by the prototypic hypovirus CHV1-EP713 include reduced orange pigmentation, reduced asexual sporulation, and loss of female fertility (1, 2, 4, 21, 22).The multiple phenotypic changes associated with hypovirus infection are accompanied by changes in the expression of specific cellular genes, e.g., the genes for laccase (16, 45), a sexual pheromone (54), the cell wall hydrophobin cryparin (56), a cellulobiohydrolase (53), a cutinase (52), and a polygalacturonase (23, 26). Using differential display, Chen et al. (12) provided evidence that hypovirus infection causes a rather extensive alteration in the host gene expression profile; more than 40...