Rescue of Defective Poliovirus RNA Replication by 3AB-Containing Precursor Polyproteins

Poliovirus (PV), the prototypic picornavirus, replicates its genomic RNA via membranous replication complexes within the cytoplasm of an infected cell (11, 12). These complexes appear as rosette-like structures (8) and are thought to provide an environment for increased local concentrations of poliovirus proteins, presumably limiting diffusion within the replication complex (22). Highly specific interactions between cellular and viral proteins associated with virion RNA (vRNA) and cellular membranes result in the formation of PV replication complexes. These interactions include tight membrane-protein associations by PV proteins 3AB (17, 40, 45, 47, 52) and 2C (15, 20, 48) combined with specific RNA-protein interactions between the PV 5′ noncoding region (5′NCR) and the viral polypeptide 3CD (2, 3). In addition, the presence of the 5′ terminal ∼100 nucleotides (nt) of PV RNA mediates an in vitro interaction between 3CD and the cellular protein poly(rC) binding protein 2 (21, 36). The viral polypeptide 3AB (24, 54) and the cellular protein EF-1α (24) also appear to complex with 3CD and the 5′ end of the PV genome. Interactions between the viral proteins 3D and 3AB have been documented (25, 29, 37), as have RNA-protein interactions involving the 3′ ends of positive- and negative-strand picornavirus RNAs (4, 39, 50). While many of these studies have focused on individual molecular interactions, little is known about the viral polyprotein subunits necessary for the initial assembly of the vRNA replication complex. For PV, like all picornaviruses, mature gene products are specifically processed from viral precursor polyproteins (42). The efficient and highly regulated protein processing cascade produces cleavage products with functions distinct from those of their precursor proteins. Due to the short half-lives of large precursor proteins, it has been difficult to determine the composition of assembly intermediates. An additional control of RNA replication relies on the translation of PV RNA; that is, translation of a particular genome is a prerequisite for that genome to be competent for replication due to the requirement for either a cis-acting viral protein(s) or ribosomal passage over the RNA template (34). A relationship between ribosomal passage and RNA replication stems from the observation that all naturally occurring defective interfering particles contain deletions in the capsid region (P1 [Fig. 1]) that maintain the reading frame (28). The requirement for translation prior to RNA replication was later confirmed by using genetically engineered replicons in which replication occurred only if deletions were in frame and the P2-P3 region was left intact (except for the N terminus of viral protein 2A) (16). The apparent cis dominance of translation over replication does not in theory preclude the utilization of some of the viral nonstructural proteins in trans. For example, passage of the ribosome could transiently expose a cis-acting replication determinant present on the RNA template, while the active replication proteins in the complex could consist of mixed viral proteins, some of which were synthesized from other viral mRNAs. FIG. 1 Schematic representation of full-length and subgenomic PV RNAs. (A) Schematic representation of the PV genomic structure and viral protein organization where the viral structural proteins are derived from the P1 portion of the genome and the nonstructural ... Complementation of site-specific lesions in trans has been assayed by providing the wild-type gene product(s) through a helper virus (6, 7, 14, 18, 22, 26, 32, 49) and through novel approaches using dicistronic RNAs or amber-suppressing cell lines (13, 34). The collective results of such studies indicate that each viral protein likely has multiple functions, some of which can be complemented in trans and some of which cannot, depending on whether a mutation exerts its effect at the level of the precursor protein or at the level of the mature cleavage product. Previous experimental approaches often utilized a helper virus or helper RNA to provide the complementing gene products in infected or transfected cells. vRNAs produced in cells from such approaches most likely generate individual RNA replication complexes that are physically separated from each other in the cytoplasm. By the time viral proteins are generated to levels sufficient for diffusion and effective complementation, polypeptides in the replication complexes may be processed or assembled such that they are no longer competent for subunit exchange. In this study, we attempted to circumvent the problem of inaccessibility to the PV replication complex by using a HeLa cell-free replication assay to produce potential complementing viral proteins during the initial stages of complex formation. We addressed four questions. (i) What polyprotein subunit delivers the RNA replication function of viral protein 3AB during assembly of the replication complex? (ii) Can this 3AB-containing assembly intermediate by provided in trans? (iii) Does this assembly subunit need to be proteolytically active? (iv) Does 3AB need to be physically linked to the active viral RNA polymerase during complex assembly? Our results show that a 3AB mutation causing a severe RNA replication defect can be efficiently complemented in trans by providing the large replicase precursor (P3) but not the mature 3AB polypeptide. Furthermore, the rescuing trans-P3 protein must contain an active 3C proteolytic domain but not an active polymerase domain. Possible mechanisms for complementation are discussed.