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Journal of Virology, May 1999, p. 3854-3865, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Selection of RNA Replicons Capable of Persistent Noncytopathic Replication in Mammalian Cells

Ilya Frolov, Eugene Agapov, Thomas A. Hoffman Jr., Béla M. Prágai,dagger Mara Lippa, Sondra Schlesinger, and Charles M. Rice*

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093

Received 1 October 1998/Accepted 19 January 1999

The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves transmission to vertebrate hosts via mosquitoes. Chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mRNA translation and cell death. Using engineered Sindbis virus RNA replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing persistent, noncytopathic replication in BHK-21 cells. Two of these adaptive mutations involved single-amino-acid substitutions in the C-terminal portion of nsP2, the viral helicase-protease. At one of these loci, nsP2 position 726, numerous substitution mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity. This work also provides a series of alphavirus replicons for noncytopathic gene expression studies (E. V. Agapov, I. Frolov, B. D. Lindenbach, B. M. Prágai, S. Schlesinger, and C. M. Rice, Proc. Natl. Acad. Sci. USA 95:12989-12994, 1998) and a general strategy for selecting RNA viral mutants adapted to different cellular environments.

* Corresponding author. Mailing address: Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110-1093. Phone: (314) 362-2842. Fax: (314) 362-1232. E-mail: rice{at}borcim.wustl.edu.

dagger Permanent address: Department of Microbiology, Albert Szent-Györgyi Medical University, Szeged, Hungary.

Journal of Virology, May 1999, p. 3854-3865, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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