Based upon the assumption that the 3′ NCR of poliovirus acts as a cis-
acting signal, Todd et al. had previously attempted to define the minimal element necessary for viral genome replication (51
). We deleted the entire genomic 3′ NCR and found that this mutant virus replicated to within 1 log10
unit of wild-type virus levels in HeLa cells (51
). This finding was surprising, because the 3′ NCR of the three poliovirus serotypes is well conserved despite the fact that the viral RNA-dependent RNA polymerase has a high error rate (12
). To define a biologically relevant role for the 3′ NCR, we examined the growth of the Δ3′ NCR virus in different cell types. It has been demonstrated that a point mutation in the poliovirus 5′ NCR reduces neurovirulence in mice and causes a replication defect in neuroblastoma tissue culture cells (3
). In this report, we showed that Δ3′ NCR PV1 accumulates more slowly and to a lower titer in a neuroblastoma cell line (SK-N-SH) than in HeLa cells as a result of an exacerbated RNA replication defect. The phenotype is not likely due to a cell-dependent translation deficiency, because the virus translation efficiency was decreased only moderately. It is likely that the decrease in translation efficiency in SK-N-SH cells is a by-product of the reduced number of RNA genomes caused by the defect in viral genome replication, although we cannot rule out the contribution of a slight defect in translation to reduced levels of nonstructural proteins available for viral RNA replication functions.
Neurovirulence of the deletion mutant virus was markedly attenuated in PVR transgenic mice. Compared to wild-type virus, greater than 1,000 times more mutant virus was required to induce paralysis following IC inoculations. The level of attenuation of the deletion mutant was comparable to that of the Sabin type 1 vaccine strain. The PD50
of Δ3′ NCR PV1 was ~3 × 107
PFU, and the PD50
of Sabin 1 in the same PVR transgenic mouse line has been reported to be >5 × 107
). Although it is possible that the attenuation phenotype seen in the PVR transgenic mice is the result of a species-specific requirement for the 3′ NCR, the PD50
was in good agreement with the growth characteristics of the mutant virus in the human SK-N-SH cell line. None of the nontransgenic mice showed symptoms of infection when inoculated with high-titer Δ3′ NCR PV1 stocks, allowing us to conclude that the symptoms induced in PVR transgenic mice infected with mutant virus were due to the ability of the transgenic mice to support mutant virus replication. It will be interesting to determine whether deletion mutant virus recovered from the transgenic mice has accumulated any additional mutations that allow higher levels of replication compared to those of input Δ3′ NCR virus. In a related study, Merkle and colleagues recently observed that a mutant coxsackievirus B3 harboring a partial deletion within the 3′ NCR of genomic RNA was attenuated for induction of myocarditis in infected mice, suggesting a role for the coxsackievirus 3′ NCR in viral pathogenesis (36
It has been reported that the ratio of positive- to negative-strand RNA accumulation is lower in leukocytic and nerve cells following infections with wild-type poliovirus (31
). We report a similar finding here, where the ratio of positive- to negative-strand RNA was lower in neuronal cells infected with wild-type virus than in infected HeLa cells. These data suggest that there is a cell-specific contribution to positive-strand RNA synthesis and that a cellular factor(s) involved in positive-strand RNA synthesis is limiting in cells of neuronal origin. After initiation of negative-strand RNA synthesis occurs, the mutant virus faces an exacerbated strand-specific defect in positive-strand RNA synthesis. This was evidenced by the lower ratio of positive-strand to negative-strand viral RNA during an infection with the mutant virus compared to the ratio detected during a wild-type poliovirus infection. In order to observe such a phenotype, the mutant virus would either have to synthesize higher levels of negative-strand RNAs or lower levels of positive-strand RNAs during genome replication. The data shown in Fig. do not reveal significant differences in the levels of negative-strand RNAs found in cells infected with wild-type or Δ3′ NCR PV1. In addition, the results shown in Fig. indicate that we did not detect any differences in stability of positive-strand viral RNAs during infection of neuronal cells by wild-type or mutant virus in the absence of viral RNA synthesis. Therefore, it is likely that the lower ratio of positive- to negative-strand RNA is due to reduced levels of positive-strand RNA synthesis.
We found that the strand-specific defect in Δ3′ NCR mutant RNA synthesis was exacerbated in neuronal cells, suggesting that a cellular factor(s) involved in positive-strand RNA synthesis interacts with the poliovirus 3′ NCR during genomic replication. The differences in phenotypic growth patterns may be the result of a lower concentration, posttranslational modification, alternative splicing patterns, or alternative patterns of subcellular sequestering of a host factor(s). Alternatively, the mutant virus growth defect could be due to differences in cellular processes, such as intracellular motility, membranous vesicle trafficking, or differences in cell morphology that could exacerbate a defect caused by a lack of genomic tethering to the replication complexes.
The observation that an RNA structure located at one terminus of the poliovirus genome functions in initiation of RNA synthesis at the opposite terminus is not without precedent. There is evidence that the 5′-terminal cloverleaf structure (also known as stem-loop I) located on the positive strand of poliovirus genomic RNA is involved in initiation of negative-strand RNA synthesis (6
). Thus, it is possible that a similar mechanism of 5′-3′ termini interaction or communication is required for positive-strand RNA synthesis and that this putative interaction is provided, in part, by the genomic 3′ NCR. We have not yet determined whether these RNA sequences or secondary structures that are necessary for the initiation of positive-strand synthesis function in the context of the 3′ ends of the positive strands, acting in trans
, or in the 5′ ends of negative-strand intermediates, acting in cis
. Independent of the precise mechanism of attenuation caused by the strand-specific defect in RNA synthesis, the cell-dependent replication phenotype of the deletion mutant virus reveals a previously unknown property of the poliovirus 3′ NCR.