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Journal of Virology, November 2002, p. 10776-10784, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10776-10784.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

Bin Lu, Chien-Hui Ma, Robert Brazas,{dagger} and Hong Jin*

Medimmune Vaccines, Inc., Mountain View, California 94043

Received 2 May 2002/ Accepted 30 July 2002

The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of 33Pi-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.

* Corresponding author. Mailing address: Medimmune Vaccines, Inc., 297 N. Bernardo Ave., Mountain View, CA 94043. Phone: (650) 919-6587. Fax: (650) 919-2418. E-mail: jinh{at}medimmune.com.

{dagger} Present address: Department of Genetics, Duke University Medical Center, Durham, NC 27710.

Journal of Virology, November 2002, p. 10776-10784, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10776-10784.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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