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Journal of Virology, October 2003, p. 10575-10583, Vol. 77, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.19.10575-10583.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Exploitation of Nucleic Acid Packaging Signals To Generate a Novel Influenza Virus-Based Vector Stably Expressing Two Foreign Genes

Tokiko Watanabe,1,2,3 Shinji Watanabe,3 Takeshi Noda,1,4 Yutaka Fujii,1,5 and Yoshihiro Kawaoka1,2,3*

Institute of Medical Science, University of Tokyo, Tokyo 108-8639,1 CREST, Japan Science and Technology Corp., Saitama 332-0012,2 Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818,4 Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan,5 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 537063

Received 2 May 2003/ Accepted 9 July 2003

At the final step in viral replication, the viral genome must be incorporated into progeny virions, yet the genomic regions required for this process are largely unknown in RNA viruses, including influenza virus. Recently, it was reported that both ends of the neuraminidase (NA) coding region are critically important for incorporation of this vRNA segment into influenza virions (Y. Fujii, H. Goto, T. Watanabe, T. Yoshida, and Y. Kawaoka, Proc. Natl. Acad. Sci. USA 100:2002-2007, 2003). To determine the signals in the hemagglutinin (HA) vRNA required for its virion incorporation, we made a series of deletion constructs of this segment. Subsequent analysis showed that 9 nucleotides at the 3' end of the coding region and 80 nucleotides at the 5' end are sufficient for efficient virion incorporation of the HA vRNA. The utility of this information for stable expression of foreign genes in influenza viruses was assessed by generating a virus whose HA and NA vRNA coding regions were replaced with those of vesicular stomatitis virus glycoprotein (VSVG) and green fluorescent protein (GFP), respectively, while retaining virion incorporation signals for these segments. Despite the lack of HA and NA proteins, the resultant virus, which possessed only VSVG on the virion surface, was viable and produced GFP-expressing plaques in cells even after repeated passages, demonstrating that two foreign genes can be incorporated and maintained stably in influenza A virus. These findings could serve as a model for the construction of influenza A viruses designed to express and/or deliver foreign genes.

* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Dr., Madison, WI 53706. Phone: (608) 265-4925. Fax: (608) 265-5622. E-mail: kawaokay{at}svm.vetmed.wisc.edu.

Journal of Virology, October 2003, p. 10575-10583, Vol. 77, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.19.10575-10583.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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