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Journal of Virology, April 2001, p. 3841-3850, Vol. 75, No. 8
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.8.3841-3850.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Requirements for Assembly of Poliovirus Replication Complexes and Negative-Strand RNA Synthesis

Natalya L. Teterina,1 Denise Egger,2 Kurt Bienz,2 David M. Brown,3 Bert L. Semler,3 and Ellie Ehrenfeld1,*

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland1; Institute for Medical Microbiology, University of Basel, Basel, Switzerland2; and Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California3

Received 14 August 2000/Accepted 10 January 2001

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.

* Corresponding author. Mailing address: Laboratory of Viral Diseases, NIAID-NIH, Building 9, Room 1 E 100, MSC 0930, Bethesda, MD 20892-0930. Phone: (301) 435-1114. Fax: (301) 435-6021. E-mail: Ehrenfee{at}csr.nih.gov.

Journal of Virology, April 2001, p. 3841-3850, Vol. 75, No. 8
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.8.3841-3850.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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