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Journal of Virology, August 1999, p. 6257-6264, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus

Yu-Shiu Chang,1 Ching-Len Liao,2 Chang-Huei Tsao,2 Mei-Chieh Chen,3,dagger Chiu-I Liu,3 Li-Kuang Chen,4 and Yi-Ling Lin1,2,3,*

Institute of Biomedical Sciences, Academia Sinica,1 Department of Microbiology and Immunology2 and Institute of Preventive Medicine,3 National Defense Medical Center, Taipei, and Department of Immunology, Buddhist Tzu-Chi Medical College, Hualien,4 Taiwan, Republic of China

Received 3 February 1999/Accepted 21 April 1999

Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or alpha -sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducible Escherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or beta -galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.

* Corresponding author. Mailing address: Institute of Biomedical Sciences, Academia Sinica, 128 Yen-Jiou Yuan Rd., Sec. 2, Taipei 11529, Taiwan, Republic of China. Phone: (886)-2-2652-3902. Fax: (886)-2-2782-9224. E-mail: yll{at}ibms.sinica.edu.tw.

dagger Present address: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China.

Journal of Virology, August 1999, p. 6257-6264, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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