Recombination of Poliovirus RNA Proceeds in Mixed Replication Complexes Originating from Distinct Replication Start Sites

Abstract

Genetic recombination occurs frequently during replication of picornaviruses. To explore the intracellular site and structures involved in recombination, HeLa cells were infected with poliovirus type 1 Mahoney and type 2 Sabin. The two genomes were located by fluorescent in situ hybridization and confocal microscopy. For hybridization, type-specific fluorescent riboprobes were used to visualize the same genomic region where, in parallel, recombination was demonstrated with type-specific reverse transcription-PCR and sequencing. The hybridization analysis indicated that >85% of the replication complexes contained both type 1 and type 2 RNA sequences aligned at a lateral distance of 50 nm or less. Sequential infection of cells ruled out the possibility that the high percentage of mixed replication complexes was due to aggregation of input virus. Visualization of input genomic RNA over time showed that the viral genomes migrated to relatively few distinct, and thus presumably specific, perinuclear sites where replication started. The first recombinant RNA strands could be detected concomitantly with the onset of RNA replication. The limited number of start sites for replication may be the reason for the observed preferential formation of mixed replication complexes, each accommodating several parental RNA strands and thus allowing recombination.Poliovirus (PV), the prototype member of the family Picornaviridae, represents in many respects the paradigm for research on other plus-strand RNA viruses. Among the fundamental insights gained by PV research is the first discovery of recombination between viral RNA genomes (30, 35). Since then, some light has been thrown on mechanistic aspects of recombination (for a review see references 1 and 57); however, it is not clear in which context of the elaborate structures comprising the viral RNA replication machinery, i.e., the replication complex (4,6,9,12,21,22,51,52), recombination can occur.PV RNA replication is preceded by translation of the incoming viral genome. Translation, initiated at the internal ribosomal entry site (IRES) (42, 43), was reported to be enhanced by binding of the cellular poly(rC) binding protein (PCBP) to the IRES (10,11,25) and, additionally at early stages, by an interaction of eIF4G with the IRES and the poly(A)-bound poly(A) binding protein (PABP) (50). A transition from translation to transcription was thought to be mediated by the newly synthesized viral protein 3CD, which, together with PCBP, binds to the 5Ј cloverleaf of the viral mRNA (23,24,39). Poly(A)-bound PABP then would attach to this complex, leading (again) to a 5Ј-3Ј interaction in the viral plus-strand RNA (29). This interaction was proposed to be a prerequisite for minus-strand RNA synthesis, the first step in genome replication (3,29,53). The second step in viral genome replication is plus-strand RNA synthesis, which proceeds, after multiple initiations, in the partially doublestranded replicative intermediate. The primer for RNA synthesis is VPgpUpU (41), which was found to be ...

Inducible Yeast System for Viral RNA Recombination Reveals Requirement for an RNA Replication Signal on Both Parental RNAs

“…Similar compartments are associated with RNA replication by many other positive-strand RNA viruses (8,38). BMV negative-strand RNAs are synthesized and retained in these compartments (38), consistent with observa-that the natural cowpea chlorotic mottle virus RNA3 5Ј end might have been generated by processes similar to the derivation of sURA3 RNAs from B3⌬5Ј (2).…”

“…RNA recombination events most commonly have been suggested to occur via template-switching mechanisms during RNA replication (22,24), but substantial evidence also exists for primer alignment and extension (34) or breakage-rejoining (13,14) mechanisms. Such alternate mechanisms are not necessarily mutually exclusive, as indicated by evidence for poliovirus RNA recombination by template switching (8,22), primer alignment and extension (34), and breakage-rejoining (14) mechanisms. Despite extensive studies of some specific classes of RNA recombination events, substantial uncertainties exist about the relative activity of different RNA recombination pathways, the viral and cellular determinants of their activity, and their possible relationships to normal viral replication processes.…”

Evidence for Frequent Recombination within SpeciesHuman Enterovirus BBased on Complete Genomic Sequences of All Thirty-Seven Serotypes

“…That is, there is no systematic cooperativity between crossovers at multiple sites, and the probability of multiple crossovers is generally the product of the probabilities of the individual events. An enterovirus replication complex may contain many template RNA molecules, providing opportunities for interaction between genetically distinct RNAs if the cell is coinfected with two different viruses (16,40). It has been proposed that the accumulation of RNA during replication may lead to an increase in recombination frequency due to an increase in the local concentration of donor and acceptor RNA molecules (27).…”

Role of recombination in evolution of enteroviruses

“…Thus, naturally occurring recombination can be viewed as a reliable species criterion, facilitating definition of species, which has already been applied to demonstrate the affiliation of polioviruses to the HEV-C species [49]. It is not possible to state which factors inhibit interspecies recombination: low sequence homology between different species [23], incompatibility of viral proteins or regulatory RNA elements of different enterovirus species [67][68][69], replication of different enterovirus species in diverse intracellular locations (enteroviruses were shown to replicate in few distinct replication complexes in the cell [70]), or absence of natural coreplication of different species in the same cells. In any case, the barrier to interspecies recombination has to be very strict, as no such natural recombinants have been reported so far, despite really extensive intraspecies recombination.…”