Nonreplicative RNA recombination in poliovirus

Abstract

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these "hot spots" could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5' partner, if its 3' end was oxidized or phosphorylated prior to being mixed with the 3' partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2',3'-cyclic phosphate and 5'-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.

FIG. 1

Schematic representation of the recombination partners. Solid lines correspond to segments of the poliovirus genome; the black bar denotes the inverted segment of the viral RNA (its coordinates are shown as n′). Cryptic (position 586) and initiator (position 743) AUG triplets are marked by open and solid stars, respectively. The intact and mutated (i.e., containing an oligoadenylate replacement) oligopyrimidine moieties of the essential OAT element are shown as solid and open diamonds, respectively. The silent marker mutations introduced into the 5′ partners at positions 451 and 552 are indicated by black dots. The borders of the essential replicative (oriL) and translational (IRES) elements are given on the scheme of the 5′ partners. The coordinates of the 3′-terminal nucleotides and the sequences of nonviral oligonucleotides fused to the 3′ ends of the 5′ partners, as well as the segments deleted in different 3′ partners, are given in the tables. For other details, see the text.

FIG. 2

The location of crossover sites on the 5′ partners (A) and 3′ partners (B). The composite crossover maps showing the results obtained with different combinations of the partners are presented, but the crossovers downstream of position 648 are given separately for each of the three 5′ partners. The crossover sites are denoted either by vertical bars (when the mapping was possible with a single-nucleotide accuracy) or triangles (when there was a short oligonucleotide identity in the recombining regions of the two partners). The nonviral (oligo)nucleotides found between the bodies of the 5′ and 3′ partners are shown to the right of the vertical bars corresponding to the terminal nucleotides in panel A. The position of the crossover of recombinant o22 on the 3′ partner (nt 203 to 204) lies outside the genomic region shown. The UGAAA sequence, a component of the putative cryptic hammerhead ribozyme, is shadowed. The regions of identity in the 5′ and 3′ partners (positions 568 to 634) are in boldface. The nucleotides in the inverted regions of the 3′ partners (positions 635 to 669 in ΔBB and positions 635 to 727 in the PA2 and ΔL partners) are denoted as n′. The regions of the 3′ partners supposed to form heteroduplexes with the 5′ partners are overlined (the longer overline corresponds to BG, whereas the shorter one corresponds to BN and BY partners). The recombinants obtained with oxidized (as well as oxidized and aniline-treated) or ligated with pCp 5′ partners are marked by the prefixes o and p, respectively. The crosses between individual partners yielded the following recombinants: BG × PA2, 1, 3, 5, 8, 15, 21, 25, 31, 32, 35, 36, o3, o4, and o17 to o19; BG × ΔBB, 26, 30, 33, 34, 37, and 38; BG × ΔL, o16; BN × PA2, 2, 4, 6, 10, 11, 17, 22, 23, 27, 28, o1, o2, o5, o6, o8, o10, o13, o24, and o27; BN × ΔBB, 7, 14, 18, 19, 29, and o22; BN × ΔL, 9, 12, 13, o7, o9, o11, o12, o14, o15, o23, o25, o26, o28 to o33, and p1 to p19; BY × PA2, 24 and o20; BY × ΔBB, 20, 39, and 40; BY × ΔL, 16, and o21.

FIG. 3

A model invoking the involvement of a hammerhead-like ribozyme activity in the origin of a specific hot spot of crossover sites on the 5′ partners. (A) The conserved nucleotides (boxed) and the consensus structure of hammerhead ribozymes. R, Y, and H represent a purine, pyrimidine, or any nucleotide except G, respectively. The cleavage site is shown by the arrow. (B) A hypothetical folding of the 5′ partner, generating a consensus hammerhead ribozyme annealed to a segment of the 3′ partner.

FIG. 4

A model explaining incorporation of the full-length 5′ partner into the recombinant genomes. Foreign insertions, assumed to be added by T7 RNA polymerase, are shaded. Putative reacting nucleotides are boxed. The 3′-terminal nucleotide of the 5′ partner is assumed to possess the 2′,3′-cyclic phosphate group.