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Journal of Virology, September 2005, p. 11705-11715, Vol. 79, No. 18
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.18.11705-11715.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Sialidase Activity of Influenza A Virus in an Endocytic Pathway Enhances Viral Replication

Takashi Suzuki,^1,2,* Tadanobu Takahashi,^1,2, Chao-Tan Guo,^1,2,3 Kazuya I.-P. Jwa Hidari,^1,2 Daisei Miyamoto,^1,2 Hideo Goto,^2,4 Yoshihiro Kawaoka,^2,4,5,6 and Yasuo Suzuki^1,2*

Department of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences and COE Program in the 21st Century, Shizuoka, Japan,¹ CREST, Japan Science and Technology Agency, Saitama, Japan,² Institute of Bioengineering, Zhejiang Academy of Medical Sciences, Hang Zhou, China,³ Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo,⁴ International Research Center for Infectious Diseases, Tokyo, Japan,⁵ Department of Pathobiological Sciences, University of Wisconsin—Madison, Madison, Wisconsin⁶

Received 10 March 2005/ Accepted 20 June 2005

N2 neuraminidase (NA) genes of the 1957 and 1968 pandemic influenza virus strains possessed avian-like low-pH stability of sialidase activity, unlike most epidemic strains. We generated four reverse-genetics viruses from a genetic background of A/WSN/33 (H1N1) that included parental N2 NAs of 1968 pandemic (H3N2) and epidemic (H2N2) strains or their counterpart N2 NAs in which the low-pH stability of the sialidase activity was changed by substitutions of one or two amino acid residues. We found that the transfectant viruses bearing low-pH-stable sialidase (WSN/Stable-NAs) showed 25- to 80-times-greater ability to replicate in Madin-Darby canine kidney (MDCK) cells than did the transfectant viruses bearing low-pH-unstable sialidase (WSN/Unstable-NAs). Enzymatic activities of WSN/Stable-NAs were detected in endosomes of MDCK cells after 90 min of virus internalization by in situ fluorescent detection with 5-bromo-4-chloro-indole-3-yl--N-acetylneuraminic acid and Fast Red Violet LB. Inhibition of sialidase activity of WSN/Stable-NAs on the endocytic pathway by pretreatment with 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid (zanamivir) resulted in a significant decrease in progeny viruses. In contrast, the enzymatic activities of WSN/Unstable-NAs, the replication of which had no effect on pretreatment with zanamivir, were undetectable in cells under the same conditions. Hemadsorption assays of transfectant-virus-infected cells revealed that the low-pH stability of the sialidase had no effect on the process of removal of sialic acid from hemagglutinin in the Golgi regions. Moreover, high titers of viruses were recovered from the lungs of mice infected with WSN/Stable-NAs on day 3 after intranasal inoculation, but WSN/Unstable-NAs were cleared from the lungs of the mice. These results indicate that sialidase activity in late endosome/lysosome traffic enhances influenza A virus replication.

These authors contributed equally to this work.