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Journal of Virology, December 2003, p. 13106-13116, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13106-13116.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Functional Analysis of nsP3 Phosphoprotein Mutants of Sindbis Virus

Indra Dé,{dagger} Cori Fata-Hartley,{ddagger} Stanley G. Sawicki, and Dorothea L. Sawicki*

Department of Microbiology, Medical College of Ohio, Toledo, Ohio 43699

Received 17 June 2003/ Accepted 15 September 2003

Alphavirus nsP3 phosphoprotein is essential for virus replication and functions initially within polyprotein P123 or P23 components of the short-lived minus-strand replicase, and upon polyprotein cleavage, mature nsP3 likely functions also in plus-strand synthesis. We report the identification of a second nsP3 mutant from among the A complementation group of Sindbis virus (SIN) heat-resistant strain, ts RNA-negative mutants. The ts138 mutant possessed a change of G4303 to C, predicting an Ala68-to-Gly alteration that altered a conserved His-Ala-Val tripeptide in the ancient (pre-eukaryotic), "X" or histone 2A phosphoesterase-like macrodomain that in SIN encompasses nsP3 residues 1 to 161 and whose role is unknown. We undertook comparative analysis of three nsP3 N-terminal region mutants and observed (i) that nsP3 and nsP2 functioned initially as a single unit as deduced from complementation analysis and in agreement with our previous studies, (ii) that the degree of phosphorylation varied among the nsP3 mutants, and (iii) that reduced phosphorylation of nsP3 correlated with reduced minus-strand synthesis. The most striking phenotype was exhibited by ts4 (Ala268 to Val), which after shift to 40°C made significantly underphosphorylated P23/nsP3 and lost selectively the ability to make minus strands. After shift to 40°C, mutant ts7 (Phe312 to Ser) made phosphorylated P23/nsP3 and minus strands but failed to increase plus-strand synthesis. Macrodomain mutant ts138 was intermediate, making at 40°C partially phosphorylated P23/nsP3 and reduced amounts of minus strands. The mutants were able to assemble their nsPs at 40°C into complexes that were membrane associated. Our analyses argue that P23/P123 phosphorylation is affected by macrodomain and Ala268 region sequences and in turn affects the efficient transcription of the alphavirus genome.

* Corresponding author. Mailing address: Department of Microbiology, Medical College of Ohio, Toledo, OH 43699. Phone: (419) 383-4337. Fax: (419) 383-3002. E-mail: dsawicki{at}mco.edu.

{dagger} Present address: Infectious Diseases, Department of Medicine, M. D. Anderson, University of Texas, Houston, Tex.

{ddagger} Present address: Department of Biochemistry, Institute of Molecular Virology, University of Wisconsin—Madison, Madison, Wis.

Journal of Virology, December 2003, p. 13106-13116, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13106-13116.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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