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Journal of Virology, July 2000, p. 5880-5885, Vol. 74, No. 13
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Cys3-His1 Motif of the Respiratory Syncytial Virus M2-1 Protein Is Essential for Protein Function

Richard W. Hardydagger and Gail W. Wertz*

Department of Microbiology, University of Alabama School of Medicine at Birmingham, Birmingham, Alabama 35294

Received 10 February 2000/Accepted 6 April 2000

The M2 gene of respiratory syncytial (RS) virus has two open reading frames (ORFs). ORF1 encodes a 22-kDa protein termed M2-1. The M2-1 protein contains a Cys3-His1 motif (C-X7-C-X5-C-X3-H) near the amino terminus. This motif is conserved in all human, bovine, and ovine strains of RS virus. A similar motif found in the mammalian transcription factor Nup475 has been shown to bind zinc. The M2-1 protein of human RS virus functions as a transcription factor which increases polymerase processivity, and it enhances readthrough of intergenic junctions during RS virus transcription, thereby acting as a transcription antiterminator. The M2-1 protein also interacts with the nucleocapsid protein. We examined the effects of mutations of cysteine and histidine residues predicted to coordinate zinc in the Cys3-His1 motif on transcription antitermination and N protein binding. We found that mutating the predicted zinc-coordinating residues, the cysteine residues at amino acid positions 7 and 15 and the histidine residue at position 25, prevented M2-1 from enhancing transcriptional readthrough. In contrast, mutations of amino acids within this motif not predicted to coordinate zinc had no effect. Mutations of the predicted zinc-coordinating residues in the Cys3-His1 motif also prevented M2-1 from interacting with the nucleocapsid protein. One mutation of a noncoordinating residue in the motif which did not affect readthrough during transcription, E10G, prevented interaction with the nucleocapsid protein. This suggests that M2-1 does not require interaction with the nucleocapsid protein in order to function during transcription. Analysis of the M2-1 protein in reducing sodium dodecyl sulfate-polyacrylamide gels revealed two major forms distinguished by their mobilities. The slower migrating form was shown to be phosphorylated, whereas the faster migrating form was not. Mutations in the Cys3-His1 motif caused a change in distribution of the M2-1 protein from the slower to the faster migrating form. The data presented here show that the Cys3-His1 motif of M2-1 is essential for maintaining the functional integrity of the protein.

* Corresponding author. Mailing address: Department of Microbiology, University of Alabama School of Medicine at Birmingham, BBRB Box 17, Room 366, 845 19th St. South, Birmingham, AL 35294. Phone: (205) 934-0877. Fax: (205) 934-1636. E-mail: gail_wertz{at}microbio.uab.edu.

dagger Present address: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.

Journal of Virology, July 2000, p. 5880-5885, Vol. 74, No. 13
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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