Internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants.

Abstract

Neuropathogenicity of poliovirus can be attenuated by mutations in the internal ribosomal entry site (IRES) within the 5' nontranslated region of its genome. The Sabin vaccine strains used in prevention of poliomyelitis carry such mutations in their IRES elements. In addition, mutations within the structural and nonstructural proteins of Sabin strains may equally contribute to the attenuation phenotype. Despite their effectiveness as vaccines, the Sabin strains retain a neuropathogenic potential in animal models for poliomyelitis and, at a very low rate, they can cause poliomyelitis in vaccine recipients. The elimination of the neurocytopathic phenotype was achieved through the exchange of the entire poliovirus IRES with its counterpart from human rhinovirus type 2 without affecting growth properties in nonneuronal cells. The attenuating effect of the human rhinovirus type 2 IRES within the context of a poliovirus genome has been mapped to the 3' portion of this genetic element.Models for the mechanism of eukaryotic translation demand ribosomal binding at a 5' terminal cap and ribosome scanning until the proper site of initiation is recognized (1). However, efficient translation of picornavirus RNAs that lack a 5' m7GpppG cap structure (2) occurs via internal ribosomal entry within the 5' nontranslated region (5' NTR) (3-6). The presence of numerous clustered initiation codons with proper context for translational initiation within picornavirus 5' NTRs makes a scanning mechanism implausible. It was found that disproportionally long picornavirus 5' NTRs contain a cis-acting genetic element, the internal ribosomal entry site (IRES), that mediates internal ribosomal entry and capindependent translational initiation (3-8). Trans-acting cellular and viral factors interacting with the IRES, which ensure proper initiation and stimulation of translation, have been identified (9-13). Although a large number of studies on IRES function (mostly in cell-free systems) have shed some light on the mechanism of translational initiation in IRES elements (reviewed in ref.2), their function in determination of viral pathogenesis in vivo remains obscure.We have constructed picornavirus genomic hybrids in which the IRES element of poliovirus (PV) was replaced with that of either encephalomyocarditis virus (EMCV) or human rhinovirus type 2 (HRV2). Exchange of its generic IRES element affected the most distinctive pathogenic property of PV. Neuropathogenicity in a mouse model for poliomyelitis was eliminated in viruses containing the HRV2 IRES while still present in attenuated PV vaccine strains currently in use. Recombinant PV strains featuring the HRV2 IRES regained neurovirulence upon transfer of two distinctive domains derived from the PV IRES. IRES-mediated cell specificity represents a new determinant of picornaviral pathogenesis at the level of viral gene expression.

A nonpolio enterovirus with respiratory tropism causes poliomyelitis in intercellular adhesion molecule 1 transgenic mice

“…The fact that PV-infected CD155 tg mice develop poliomyelitis bearing the hallmarks of the primate disease (12)(13)(14) underscores the cardinal role of receptor preference in EV disease specificity.…”

“…Plasmid pCAV21 (strain Kuykendall) was cut with MluI and transcribed in vitro with T7 RNA polymerase (Stratagene) to derive virus, as described (14). Anesthetized mice (10-28 wk old) were injected with CAV21 [2.8 ϫ 10 8 plaque-forming units (pfu) in 30-100 l of PBS] into the gastrocnemius muscle with a 27-gauge needle.…”

“…Infected animals were monitored daily for signs of disease. Virus levels in gastrocnemius muscle (average mass, 0.135 g) and spinal cord (average mass, 0.047 g) were determined from two animals at each designated time point, as described (14). Sciatic nerve transection (16) and intracerebral stereotaxic injections (19) were carried out as described.…”

The Promoters for Human and Monkey Poliovirus Receptors

“…A very similar footprint pattern was obtained when the experiment was carried out with nuclear extracts of mouse L cells (data not shown), a result suggesting that factors controlling hPVR promoter activity and transcriptional start site selection are similar in human and mouse cells. This was not unexpected, since mice transgenic for the hPVR gene are not only sensitive to poliovirus infection, but when inoculated intravenously or intracerebrally, they produce a disease syndrome nearly identical to human poliomyelitis (24,25,41,42).…”

Characterization of in vitro and in vivo phenotypes of poliovirus type 1 mutants with reduced viral protein synthesis activity

“…These results showed that the effect of reduced viral protein synthesis activity in the range examined on the attenuation of PV was limited, hence other steps in the infection process may play major roles in the attenuation. The mutants examined in this study make a contrasting example to the RIPO mutant, which is a PV mutant with rhinovirus IRES that replicated well in HeLa cells but was not neurovirulent in PV receptor-expressing transgenic mice (Gromeier et al, 1996).…”