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Journal of Virology, January 2009, p. 29-36, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.02293-07
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Interaction of the Influenza A Virus Nucleocapsid Protein with the Viral RNA Polymerase Potentiates Unprimed Viral RNA Replication{triangledown}

Laura L. Newcomb ,1,{dagger},{ddagger} Rei-Lin Kuo,1,{ddagger} Qiaozhen Ye,2 Yunyun Jiang,2 Yizhi Jane Tao,2 and Robert M. Krug1*

Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712,1 Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 770052

Received 22 October 2007/ Accepted 10 October 2008

The influenza A virus polymerase transcribes and replicates the eight virion RNA (vRNA) segments. Transcription is initiated with capped RNA primers excised from cellular pre-mRNAs by the intrinsic endonuclease of the viral polymerase. Viral RNA replication occurs in two steps: first a full-length copy of vRNA is made, termed cRNA, and then this cRNA is copied to produce vRNA. The synthesis of cRNAs and vRNAs is initiated without a primer, in contrast to the initiation of viral mRNA synthesis, and requires the viral nucleocapsid protein (NP). The mechanism of unprimed viral RNA replication is poorly understood. To elucidate this mechanism, we used purified recombinant influenza virus polymerase complexes and NP to establish an in vitro system that catalyzes the unprimed synthesis of cRNA and vRNA using 50-nucleotide-long RNA templates. The purified viral polymerase and NP are sufficient for catalyzing this RNA synthesis without a primer, suggesting that host cell factors are not required. We used this purified in vitro replication system to demonstrate that the RNA-binding activity of NP is not required for the unprimed synthesis of cRNA and vRNA. This result rules out two models that postulate that the RNA-binding activity of NP mediates the switch from capped RNA-primed transcription to unprimed viral RNA replication. Because we showed that NP lacking RNA-binding activity binds directly to the viral polymerase, it is likely that a direct interaction between NP and the viral polymerase results in a modification of the polymerase in favor of unprimed initiation.

* Corresponding author. Mailing address: Institute for Cellular and Molecular Biology, University of Texas at Austin, 2500 Speedway, Austin, TX 78712. Phone: (512) 232-5563. Fax: (512) 232-5565. E-mail: rkrug{at}mail.utexas.edu

{triangledown} Published ahead of print on 22 October 2008.

{dagger} Present address: Biology Department, California State University San Bernardino, San Bernardino, CA 92407.

{ddagger} These two authors made equal contributions to this work.

Journal of Virology, January 2009, p. 29-36, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.02293-07
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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