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Journal of Virology, January 2009, p. 562-571, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01424-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Studies of an Influenza A Virus Temperature-Sensitive Mutant Identify a Late Role for NP in the Formation of Infectious Virions {triangledown}

Sarah L. Noton,1,{dagger} Martha Simpson-Holley,1,{ddagger} Elizabeth Medcalf,1,§ Helen M. Wise,1 Edward C. Hutchinson,1 John W. McCauley,2,¶ and Paul Digard1*

Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom,1 Institute for Animal Health, Compton Laboratory, Berkshire RG20 7NN, United Kingdom2

Received 9 July 2008/ Accepted 27 October 2008

The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner.

* Corresponding author. Mailing address: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom. Phone: 44 1223 336920. Fax: 44 1223 336926. E-mail: pd1{at}mole.bio.cam.ac.uk

{triangledown} Published ahead of print on 5 November 2008.

{dagger} Present address: Department of Microbiology, Boston University School of Medicine, Boston, MA 02115.

{ddagger} Present address: Whitehead Institute, Nine Cambridge Center, Cambridge, MA 02142.

§ Present address: Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, United Kingdom.

Present address: Division of Virology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

Journal of Virology, January 2009, p. 562-571, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01424-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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