One-step growth curves of intergeneric IRES domain recombinants shown in Fig. 1. Growth curves were established as described before (7) in SK-N-MC cells (A) and HeLa cells (B). IRES constructs with intergeneric combined domains V and VI, viruses PV1(R2-4) (+), PV1(R2-4,6) (□), PV1(R2-5) (▴), PV1(R5) (■), PV1(R5-6) (▵), and PV1(R6) (×), failed to drive efficient PV replication in SK-N-MC cells, similar to the degree of growth restriction observed with the intact HRV2 IRES [PV1(RIPO) (○)]. Extensive domain swapping barely affected the ability of the recombinant IRES elements to function efficiently in HeLa cells [compare recombinant growth curves with that for PV1(M): (•)]. p.i., postinfection.

PV1/HRV2 chimeras featuring heterologous or synthetic IRESes of various composition. The cognate IRES of PV1(M) (A) was exchanged with its counterpart from HRV2 as described previously [B; PV1(RIPO) (7)]. All viruses feature the wt PV1(M) ORF with the exception of C [PV1(RIPOS)], which was engineered to contain all mutations within the capsid encoding region (P1) specifying PV1(S). The cloverleaf (5′NTR domain I) was derived from PV1(M) in all viruses. All featured IRESes in a PV context were tested for their propensity to mediate neurological disease after intracerebral (i.c.) inoculation into CD155 tg mice (third column). A horizontal bar indicates that intracerebral inoculation of 10⁹ PFU of the variant in question did not elicit any neurological symptoms in CD155 tg mice.

Growth characteristics of PV1(rpr) (□), PV1(rpp) (■), and PV1(prr) (×) in HeLa cells and SK-N-MC cells compared to one-step growth curves of PV1(M) (•) and PV1(RIPO) (○). Growth curves established in SK-N-MC neuroblastoma cells (A) and HeLa cells (B) demonstrate cell-specific growth capacities exhibited by different IRES recombinants. All viruses replicated with equal efficiency in HeLa cells. Propagation of PV1(RIPO) in SK-N-MC cells is exceedingly poor. Replacing nucleotide sequences of PV1(M) within the upper loop regions of domains V and VI in an HRV2 background [PV1(rpp) and PV1(rpr)] restored neurovirulence, indicated by their wt growth characteristics in cells of neuronal derivation. The converse recombinant virus of PV1(rpr), containing HRV2 sequences in the uppermost regions of domain V and VI within a PV1(M) IRES [PV1(prr)], was unable to efficiently replicate in neuroblastoma cell lines, underlining the critical importance for cooperative function of stem loops V and VI in the determination of a neurovirulent phenotype of PV. p.i., postinfection.

Construction of PV1(M)/HRV2 intradomain chimeras featuring heterologous uppermost regions of domains V and VI linked to the stem structures originating from HRV2 and PV1(M), respectively. Sequences from PV1(M) are shown on a white background; structures derived from HRV2 are boxed in light gray. (A) The 5′NTR of PV1(RIPO) with heterologous distal stem loop structures V and VI derived from PV1(M) yield PV1(rpp). The nucleotide numbering of the PV1(M) IRES was applied to the corresponding HRV2 sequence. Asterisks indicate the positions of single point mutations implicated in the att phenotype of the Sabin vaccine strains (see text). A KpnI endonuclease restriction site introduced for cloning purposes is outlined by a white box. Each dark gray box delineates the position of a silent AUG triplet conserved among all enterovirus and rhinovirus strains. Recombinant IRES A [PV1(rpp)] features the authentic initiating AUG triplet of PV1(M) separated from domain VI by a 154-nt stretch. In construct B, deletion of the 154-nt spacer separating the IRES from the initiating AUG triplet in PV1(rpp) yielded PV1(rpr), creating an AUG codon within an optimal Kozak context (…AnnAUGG…) characteristic of HRV2. For construct C, a synthetic IRES converse to B [PV1(rpr)] was generated by exchanging those nucleotides within the upper stem loop regions of IRES domains V and VI of PV1(M) outlined by light gray boxes with their counterparts from HRV2, yielding PV1(prr).

Sequence alignment of IRES domains V and VI of PV1(M) (17) and HRV2 (33). GenBank accession numbers for the sequences used are J02281 for PV1(M) and X02316 for HRV2. The stem loop structures indicated by the superimposed lines are based on published secondary structure calculations, biochemical probing, computational methods, and comparative sequence analysis (21, 29, 34). Shaded areas delineate segments of homology between PV1(M) and HRV2. Note that extensive homology is present between the IRES domains of these virus species in areas forming the ascending and descending lower stems of both domains V (nt 448 to 482 and 510 to 562) and VI (nt 581 to 596), whereas the upper loop regions of domains V (nt 483 to 509) and VI (nt 597 to 606) are characterized by sequence disparity. Roman numerals indicate the major loop structures forming the tips of domains V and VI, respectively (see Fig. 4 for secondary structure). Despite a high degree of conservation of secondary structure in the IRES nt 100 to 580, the architecture of domain VI of HRV2 is different from that of PV1(M) (indicated by upper and lower domain sketches). The AUG codon initiating the HRV2 polyprotein is shown in white letters on black background. The 154-nt-long spacer region between the PV IRES and initiating AUG has been omitted (41). Sequence alignments were generated by using GeneBee-Net (version 1.0. (release 1.1) software.