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J Virol, July 1998, p. 5493-5501, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Influenza Virus Nucleoprotein Interacts with
Influenza Virus Polymerase Proteins
Siddhartha K.
Biswas,
Paul L.
Boutz, and
Debi P.
Nayak*
Department of Microbiology and Immunology,
Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los
Angeles, California 90095-1747
Received 29 January 1998/Accepted 30 March 1998
Influenza virus nucleoprotein (NP) is a critical factor in the
viral infectious cycle in switching influenza virus RNA synthesis from
transcription mode to replication mode. In this study, we investigated
the interaction of NP with the viral polymerase protein complex. Using
coimmunoprecipitation with monospecific or monoclonal antibodies, we
observed that NP interacted with the RNP-free polymerase protein
complex in influenza virus-infected cells. In addition, coexpression of
the components of the polymerase protein complex (PB1, PB2, or PA) with
NP either together or pairwise revealed that NP interacts with PB1 and
PB2 but not PA. Interaction of NP with PB1 and PB2 was confirmed by
both coimmunoprecipitation and histidine tagging of the NP-PB1 and
NP-PB2 complexes. Further, it was observed that NP-PB2 interaction was
rather labile and sensitive to dissociation in 0.1% sodium dodecyl
sulfate and that the stability of NP-PB2 interaction was regulated by
the sequences present at the COOH terminus of NP. Analysis of NP
deletion mutants revealed that at least three regions of NP interacted
independently with PB2. A detailed analysis of the COOH terminus of NP
by mutation of serine-to-alanine (SA) residues either individually or
together demonstrated that SA mutations in this region did not affect
the binding of NP to PB2. However, some SA mutations at the COOH
terminus drastically affected the functional activity of NP in an in
vivo transcription-replication assay, whereas others exhibited a
temperature-sensitive phenotype and still others had no effect on the
transcription and replication of the viral RNA. These results suggest
that a direct interaction of NP with polymerase proteins may be
involved in regulating the switch of viral RNA synthesis from
transcription to replication.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Jonsson Comprehensive Cancer Center, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1747. Phone: (310) 825-8558. Fax: (310) 206-3865. E-mail:
dnayak{at}ucla.edu.
J Virol, July 1998, p. 5493-5501, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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