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Journal of Virology, November 2005, p. 14235-14243, Vol. 79, No. 22
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.22.14235-14243.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Nidia DeJesus, David Franco, Aniko Paul, Eckard Wimmer, and Jeronimo Cello*

Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York 11794-5222

Received 3 June 2005/ Accepted 19 August 2005

The chemical synthesis of poliovirus (PV) cDNA combined with the cell-free synthesis of infectious particles yielded virus whose mouse neurovirulence was highly attenuated (J. Cello, A. V. Paul, and E. Wimmer, Science 297:1016-1018, 2002). Compared to the wild-type PV1 (Mahoney) [PV1(M)] sequence, the synthetic virus genome harbored 27 nucleotide (nt) changes deliberately introduced as genetic markers. Of the 27 nucleotide substitutions, the UA-to-GG exchanges at nucleotides 102/103, mapping to a region between the cloverleaf and the internal ribosome entry site (IRES) in the 5'-nontranslated region, were found to be involved in the observed attenuation phenotype in mice. The UA/GG mutation at nt 102/103 in the synthetic PV1(M) [sPV1(M)] background conferred also a ts phenotype of replication to the virus in human neuroblastoma cells. Conversely, the exchange of GG to wild-type (wt) UA at 102/103 in an sPV1(M) background restored wt neurovirulence in CD155 transgenic (tg) mice and suppressed the ts phenotype in SK-N-MC cells. All poliovirus variants replicated well in HeLa cells at the two temperatures, regardless of the sequence at the 102/103 locus. Analyses of variants isolated from sPV(M)-infected CD155 tg mice revealed that the G102G103-to-G102A103 reversion alone reestablished the neurovirulent phenotype. This suggests that a single mutation is responsible for the observed change of the neurovirulence phenotype. sPV1(M) RNA is translated in cell extracts of SK-N-MC cells with significantly lower efficiency than PV1(M) RNA or sPV1(M) RNA with a G102-to-A102 reversion. These studies suggest a function for the conserved nucleotide (A103) located between the cloverleaf and the IRES which is important for replication of PV in the central nervous system of CD155 tg mice and in human cells of neuronal origin.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Life Sciences Bldg., Stony Brook, NY 11794-5222. Phone: (631) 632-4432. Fax: (631) 632-8891. E-mail: jcello{at}ms.cc.sunysb.edu.

Journal of Virology, November 2005, p. 14235-14243, Vol. 79, No. 22
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.22.14235-14243.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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