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Contributions of Two Nuclear Localization Signals of Influenza A Virus Nucleoprotein to Viral Replication

Division of Virology, Department of Microbiology and Immunology,¹ International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639,⁴ Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012,² Department of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo 060-818, Japan,³ Department of Pathological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, Madison, Wisconsin 53706⁵

Received 7 July 2006/ Accepted 11 October 2006

The RNA genome of influenza A virus, which forms viral ribonucleoprotein complexes (vRNPs) with viral polymerase subunit proteins (PA, PB1, and PB2) and nucleoprotein (NP), is transcribed and replicated in the nucleus. NP, the major component of vRNPs, has at least two amino acid sequences that serve as nuclear localization signals (NLSs): an unconventional NLS (residues 3 to 13; NLS1) and a bipartite NLS (residues 198 to 216; NLS2). Although both NLSs are known to play a role in nuclear transport, their relative contributions to viral replication are poorly understood. We therefore investigated their contributions to NP subcellular/subnuclear localization, viral RNA (vRNA) transcription, and viral replication. Abolishing the unconventional NLS caused NP to localize predominantly to the cytoplasm and affected its activity in vRNA transcription. However, we were able to create a virus whose NP contained amino acid substitutions in NLS1 known to abolish its nuclear localization function, although this virus was highly attenuated. These results indicate that while the unconventional NLS is not essential for viral replication, it is necessary for efficient viral mRNA synthesis. On the other hand, the bipartite NLS, whose contribution to the nuclear transport of NP is limited, was essential for vRNA transcription and NP's nucleolar accumulation. A virus with nonfunctional NLS2 could not be generated. Thus, the bipartite NLS, but not the unconventional NLS, of NP is essential for influenza A virus replication.

Published ahead of print on 18 October 2006.

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