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Journal of Virology, January 2003, p. 1149-1156, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.1149-1156.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

An Attenuating Mutation in nsP1 of the Sindbis-Group Virus S.A.AR86 Accelerates Nonstructural Protein Processing and Up-Regulates Viral 26S RNA Synthesis

Mark T. Heise,1* Laura J. White,1 Dennis A. Simpson,1,{dagger} Christopher Leonard,1 Kristen A. Bernard,1,{ddagger} Rick B. Meeker,2 and Robert E. Johnston1

Department of Microbiology and Immunology,1 Department of Neurology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275992

Received 28 May 2002/ Accepted 8 October 2002

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Campus Box 7290, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Phone: (919) 966-4026. Fax: (919) 962-8103. E-mail: heisem{at}med.unc.edu.

{dagger} Present address: Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

{ddagger} Present address: Arbovirus Laboratories, The Wadsworth Center, The New York State Department of Health, Slingerlands, NY 12159.

Journal of Virology, January 2003, p. 1149-1156, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.1149-1156.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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