Molecular Determinants of Virulence and Stability of a Reporter-Expressing H5N1 Influenza A Virus.
We previously reported that an H5N1 virus carrying the Venus reporter gene, which was inserted into the NS gene segment from the A/Puerto Rico/8/1934(H1N1) virus (Venus-H5N1 virus), became more lethal to mice, and the reporter gene was stably maintained after mouse adaptation compared with the wild-type Venus-H5N1 (WT-Venus-H5N1) virus. However, the basis for this difference in virulence and Venus stability was unclear. Here, we investigated the molecular determinants behind this virulence and reporter stability by comparing WT-Venus-H5N1 virus with a mouse-adapted Venus-H5N1 (MA-Venus-H5N1) virus. To determine the genetic basis for these differences, we used reverse genetics to generate a series of reassortants of these two viruses. We found that reassortants with PB2 from MA-Venus-H5N1 (MA-PB2), MA-PA, or MA-NS expressed Venus more stably than did WT-Venus-H5N1 virus. We also found that a single mutation in PB2 (V25A) or in PA (R443K) increased the virulence of the WT-Venus-H5N1 virus in mice and that the presence of both of these mutations substantially enhanced the pathogenicity of the virus. Our results suggest roles for PB2 and PA in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus.


OBJECTIVE
The ability to visualize influenza viruses has far-reaching benefits in influenza virus research. Previously, we reported that an H5N1 virus bearing the Venus reporter gene became more pathogenic to mice and that its reporter gene was more highly expressed and more stably maintained after mouse adaptation. Here, we investigated the molecular determinants behind this enhanced virulence and reporter stability. We found that mutations in PB2 (V25A) and PA (R443K) play crucial roles in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus and in the virulence of influenza virus in mice. Our findings further our knowledge of the pathogenicity of influenza virus in mammals and will help advance influenza virus-related live-imaging studies in vitro and in vivo.
Exploratory Research for Advanced Technology Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo, Japan.
Dongming
D
Zhao
Exploratory Research for Advanced Technology Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo, Japan.
Satoshi
S
Fukuyama
Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo, Japan.
Shinya
S
Yamada
Exploratory Research for Advanced Technology Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan.
Tiago J S
TJ
Lopes
Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo, Japan.
Tadashi
T
Maemura
Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo, Japan.
Hiroaki
H
Katsura
Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan Transboundary Animal Distance Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
Makoto
M
Ozawa
Exploratory Research for Advanced Technology Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan Microbiology, Department of Veterinary Sciences, University of Miyazaki, Miyazaki, Japan.
Shinji
S
Watanabe
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Gabriele
G
Neumann
Exploratory Research for Advanced Technology Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo, Japan Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan kawaoka@ims.u-tokyo.ac.jp.
Yoshihiro
Y
Kawaoka
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D004285:Dogs; D005260:Female; D017930:Genes, Reporter / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000472:pathogenicity*; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008810:Mice, Inbred C57BL; D009976:Orthomyxoviridae Infections / Q000473:pathology / Q000821:virology; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D016865:Reassortant Viruses / Q000235:genetics / Q000472:pathogenicity*; D017423:Sequence Analysis, RNA; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014774:Virulence / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase