Characterization of the E-138 (Glu/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone.
A stable plasmid DNA, pMWJEAT, was constructed by using full-length Japanese encephalitis virus (JEV) cDNA isolated from the wild-type strain JEV AT31. Recombinant JEV was obtained by synthetic RNA transfection into Vero cells and designated rAT virus. JEV rAT exhibited similar large-plaque morphology and antigenicity to the parental AT31 strain. Mutant clone pMWJEAT-E138K, containing a single Glu-to-Lys mutation at aa 138 of the envelope (E) protein, was also constructed to analyse the mechanisms of viral attenuation arising from this mutation. Recombinant JEV rAT-E138K was also recovered and displayed a smaller-plaque morphology and lower neurovirulence and neuroinvasiveness than either AT31 virus or rAT virus. JEV rAT-E138K exhibited greater plaque formation than rAT virus in virus-cell interactions under acidic conditions. Heparin or heparinase III treatment inhibited binding to Vero cells more efficiently for JEV rAT-E138K than for rAT virus. Inhibition of virus-cell interactions by using wheatgerm agglutinin was more effective for JEV rAT than for rAT-E138K on Vero cells. About 20 % of macropinoendocytosis of JEV rAT for Vero cells was inhibited by cytochalasin D treatment, but no such inhibition occurred for rAT-E138K virus. Furthermore, JEV rAT was predominantly secreted from infected cells, whereas rAT-E138K was more likely to be retained in infected cells. This study demonstrates clearly that a single Glu-to-Lys mutation at aa 138 of the envelope protein affects multiple steps of the viral life cycle. These multiple changes may induce substantial attenuation of JEV.
Zhao
Zijiang
Z
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan.
Date
Tomoko
T
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan.
Li
Yuhua
Y
Chengdu Institute of Biological Products, Chengdu 610063, Sichuan Province, PR China.
Kato
Takanobu
T
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan.
Miyamoto
Michiko
M
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan.
Yasui
Kotaro
K
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan.
Wakita
Takaji
T
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan.
D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics*; D004664:Encephalitis Virus, Japanese / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D004672:Encephalitis, Japanese / Q000821:virology*; D005260:Female; D018698:Glutamic Acid / Q000235:genetics; D006493:Heparin / Q000494:pharmacology; D008239:Lysine / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D012367:RNA, Viral / Q000138:chemical synthesis / Q000235:genetics; D014162:Transfection; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics; D014774:Virulence; D014779:Virus Replication / Q000235:genetics
D018076:DNA, Complementary; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D018698:Glutamic Acid; D006493:Heparin; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase; D008239:Lysine