Mutation of a Single Conserved Nucleotide between the Cloverleaf and Internal Ribosome Entry Site Attenuates Poliovirus Neurovirulence

Abstract

Although the chemical synthesis of poliovirus (PV) cDNA combined with the cell-free synthesis of infectious particles has received much attention (7), the phenotypic properties of the synthetic virus have been largely ignored. The published sequence guiding the synthesis of the synthetic virus was that of a highly neurovirulent wild-type (wt) strain, poliovirus type 1 (Mahoney) [wt PV1(M)] (24, 43). To distinguish the synthetic virus [sPV1(M)] from PV1(M), we engineered 27 nucleotide changes into the sPV1(M) genome as genetic markers (7). Compared to the wt progenitor PV1(M) strain, the sPV1 derivative was, surprisingly, highly attenuated in transgenic mice (7) expressing the poliovirus receptor, CD155 tg mice, which have been constructed by Koike et al. (25). It was plausible that one or several of the nucleotide changes that had been introduced into the sPM1(M) genome altered the neurovirulence of sPV1(M).PV is a neurovirulent member virus of the genus Enterovirus in the family Picornaviridae. It is not yet known where the virus replicates in the gastrointestinal (GI) tract after enteric infection, but secondary lymphatic tissues most likely play a major role (21,30,33). Invasion of PV into the central nervous system is rare and altogether not necessary for viral dissemination in the population (33). Indeed, the ratio of infection to neurological complications in PV infections is very low (10 Ϫ2 to 10 Ϫ3 , depending upon the virus type). Upon invasion of the central nervous system, PV targets predominantly motor neurons for destruction, which leads to paralysis and even death (33). Only humans and nonhuman primates can be infected with PV, although humans are the only natural hosts of the virus. This host range restriction is related to CD155, the only known PV receptor (33). Construction of CD155 tg mice, however, has allowed studies of PV pathogenesis (25, 45) in these animals. We are using CD155 tg mice originally constructed by A. Nomoto and his colleagues (PVRTg21) (25). The CD155 tg mice can be infected by the intramuscular, intravenous, or intracerebral route, but they are resistant to oral infection. The reason for this restriction is that the CD155 gene is not expressed under the control of the human promoter in the GI tract of these animals (21, 61). The unexplained silence of the human CD155 promoter in the mouse GI tract prevents studies of the first crucial steps in PV enteric infection in the CD155 transgenic animals. The genome of PV, which is of plus-strand polarity, is ϳ7,441 nucleotides (nt) long. It carries a small protein, VPg, covalently attached to its 5Ј end (10,29) and is polyadenylated at its 3Ј end (60).The genome consists of a long 5Ј-nontranslated region (NTR), a single large open reading frame, and a short 3ЈNTR (24, 58). Functionally, the 5ЈNTR can be divided into two regions: the 5Ј-terminal cloverleaf (nt 1 to 89) and the internal ribosomal entry site (IRES; nt 123 to 602) (58). Polyprotein synthesis is initiated 164 nt downstream of the IRES element at nt 743 (9, 58). The clove...

Oncolytic Treatment and Cure of Neuroblastoma by a Novel Attenuated Poliovirus in a Novel Poliovirus-Susceptible Animal Model

“…The whole genome synthesis of poliovirus (14) has produced the surprising observation that a point mutation (A 103 G) in a ''spacer region'' between the cloverleaf and IRES in the 5 ¶-NTR that was introduced as genetic marker (Fig. 1A) attenuated poliovirus 10,000-fold (15). We found that the A 103 G variant of poliovirus replicates well in human neuroblastoma cell lines at 37jC (15).…”

Molecular characterization of enterovirus 71 and coxsackievirus A16 using the 5′ untranslated region and VP1 region

“…The 59UTR is relatively conserved at the nucleotide level and is often used in nucleic acid testing to diagnose enterovirus infections (Romero, 1999;Thoelen et al, 2003). However, numerous examples have shown that mutations in the 59UTR markedly decrease multiplication efficiency (Willian et al, 2000), alter cell tropism (Shiroki et al, 1997) and attenuate virulence (Tu et al, 1995;DeJesus et al, 2005). Moreover, recombination can occur in the 59UTR (Adu et al, 2007;Yang et al, 2003).…”

Oncolytic Poliovirus Therapy in a Mouse Model of Neuroblastoma: Preclinical Data Analysis and Future Studies

“…This observation prompted us to search for other poliovirus derivatives with oncolytic properties against neuroblastoma. The whole genome synthesis of poliovirus (Cello et al, 2002) produced the surprising observation that a point mutation (A103G) in a "spacer region" between the cloverleaf and IRES in the 5'-NTR that was introduced as genetic marker attenuated poliovirus 10,000-fold (De Jesus et al, 2005). We found that the A103G variant of poliovirus replicates well in human neuroblastoma cell lines at 37°C (De Jesus et al, 2005).…”

“…However, the attenuating mutation A103G in the spacer region was unstable on replication and direct revertant variants that had acquired the neurovirulent phenotype of wild-type (wt) poliovirus type 1 (Mahoney) [PV1(M)] (Fig. 1A ) were readily scored (De Jesus et al, 2005). We reasoned that a stable attenuation phenotype could be generated if the spacer region would be interrupted by an essential RNA replication element that the virus cannot afford to delete.…”

“…The whole genome synthesis of poliovirus (Cello et al, 2002) produced the surprising observation that a point mutation (A103G) in a "spacer region" between the cloverleaf and IRES in the 5'-NTR that was introduced as genetic marker attenuated poliovirus 10,000-fold (De Jesus et al, 2005). We found that the A103G variant of poliovirus replicates well in human neuroblastoma cell lines at 37°C (De Jesus et al, 2005). However, the attenuating mutation A103G in the spacer region was unstable on replication and direct revertant variants that had acquired the neurovirulent phenotype of wild-type (wt) poliovirus type 1 (Mahoney) [PV1(M)] (Fig.…”