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. 2005 Mar;79(6):3448-58.
doi: 10.1128/JVI.79.6.3448-3458.2005.

Nuclear localization of Japanese encephalitis virus core protein enhances viral replication

Yoshio Mori  1 , Tamaki Okabayashi, Tetsuo Yamashita, Zijiang Zhao, Takaji Wakita, Kotaro Yasui, Futoshi Hasebe, Masayuki Tadano, Eiji Konishi, Kohji Moriishi, Yoshiharu Matsuura
Affiliations

Nuclear localization of Japanese encephalitis virus core protein enhances viral replication

Yoshio Mori et al. J Virol. 2005 Mar.

Abstract

Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly(42) and Pro(43) in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly(42) and Pro(43) were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.

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Figures

FIG. 1.
FIG. 1.
Intracellular localization of EGFP-fused JEV core protein. Vero cells were transfected with expression plasmids encoding EGFP or EGFP-fused JEV core protein. At 24 h after transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. (A) Nuclei were stained with propidium iodide. (B) A representative nucleolar protein, nucleolin, was stained with anti-nucleolin monoclonal antibody. All samples were observed with a confocal microscope.
FIG. 2.
FIG. 2.
Role of an HCV core protein NLS in nuclear localization of JEV core protein. (A) A partial alignment of amino acid sequences of core proteins of HCV (HCVJ1 strain, genotype 1b) and flaviviruses, including JEV, SLE (St. Louis encephalitis virus), KUN, WNV, MVE (Murray Valley encephalitis virus), and DENs. Amino acid sequences of the HCV core protein and identical amino acids in flaviviruses are indicated with red letters. Amino acids that were completely conserved among the viruses are indicated by asterisks and red boldface letters. The NLS of HCV core protein previously reported by Suzuki et al. (54) is underlined. (B) Expression plasmids encoding EGFP-fused JEV core proteins mutated in the NLS of the HCV core protein. The presence (+) or absence (−) of nuclear localizations of the EGFP-fused JEV core proteins is indicated. Dots indicate the deleted amino acids. Boldface letters indicate the substituted amino acids. (C) Intracellular localization of EGFP-fused JEV core proteins with deletion or substitution in the NLS region of HCV core protein in Vero cells. Panels (except for panel f) indicate merged images of EGFP and nuclear staining by propidium iodide. Panel f shows merged images of EGFP-fused JEV core protein with a G42-to-Ala substitution and a major nucleolar protein nucleolin. All samples were observed with a confocal microscope.
FIG. 3.
FIG. 3.
Intracellular localization of core proteins in cells infected with wild-type or M4243 virus. (A) Intercellular localization of wild-type and mutant JEV. Vero or C6/36 cells were infected with wild-type or M4243 virus, fixed at 2 days postinoculation and immunostained with anti-JEV core rabbit serum. Nuclei were stained with propidium iodide. All samples were observed with a confocal microscope. (B) Intracellular fractionation of Vero cells infected with the viruses. The core proteins in the cytoplasmic and nuclear fractions were detected by Western blotting with the anti-JEV core rabbit serum. Endogenous proteins PA28α and nucleolin were detected as controls for the cytoplasmic and nuclear fractions, respectively.
FIG. 4.
FIG. 4.
Growth properties of wild-type and M4243 viruses. (A) Growth kinetics of the viruses in Vero and C6/36 cells. Both cell lines were infected with wild-type or M4243 virus at an MOI of 5. Culture supernatants were harvested at the indicated times postinoculation, and infectious titers were determined by focus-forming assays using Vero cells. Open circles and closed squares indicate the wild-type and M4243 viruses, respectively. Means of three experiments are indicated. (B) Infectious focus formation of the wild-type and M4243 viruses on Vero cells. Culture supernatants recovered at 1 or 3 days postinoculation in Vero cells were inoculated onto Vero cells and incubated for 3 days with methylcellulose overlay medium. The infectious foci were immunostained as described in Materials and Methods.
FIG. 5.
FIG. 5.
Gradient fractionation of viral particles of the wild-type and M4243 viruses. The viral particles (400 HA units) derived from Vero or C6/36 cells were applied to 10 to 40% (wt/wt) sucrose gradient and centrifuged at 147,000 × g for 90 min. Twenty fractions were collected from bottom to top and quantified by the HA test. Open circles and closed squares indicate the wild-type and M4243 viruses, respectively. The representative data from three experiments are indicated.
FIG. 6.
FIG. 6.
Viral RNA and protein syntheses in Vero and C6/36 cells infected with wild-type or M4243 virus. (A) Establishment of negative-strand JEV RNA-specific real-time RT-PCR. A series of 10-fold dilutions of synthetic negative-strand RNA in the absence (closed circles) or presence of 100-fold (gray squares) or 1,000-fold (open triangles) synthetic positive-strand RNAs were applied to the real-time RT-PCR. The Ct value represents the first PCR cycle to detect the increase in signal associated with an exponential growth of PCR product. (B) Viral negative-strand RNAs were quantified in the infected cells by the real-time RT-PCR. Open circles and closed squares indicate wild-type and M4243 viruses, respectively. The detection limit was 103 copies of viral RNA/μg of total RNA. Means of three experiments are indicated. (C) Core and NS3 proteins were detected by Western blotting with anti-JEV core rabbit serum and anti-JEV NS3 MAb 34A1, respectively. A total of 4 μg of each sample was loaded.
FIG. 7.
FIG. 7.
Complementation of M4243 replication by expression of core proteins in 293T cells. At 4 h after transfection with pCAG-GS (Vector), pCAG-WC-HA (WC-HA), or pCAG-MC-HA (Gly42 and Pro43 to Ala) (MC-HA), 293T cells were infected with wild-type or M4243 virus at an MOI of 5. At 12, 18, or 24 h postinoculation, culture supernatants and cells were harvested to apply to focus-forming assays and Western blotting or real-time RT-PCR, respectively. (A) Western blotting of 293T cells transfected with plasmids expressing HA-tagged wild-type or mutant core protein and infected with M4243 virus. Molecular mass marker was indicated in the left of the panel. (B) Growth of wild-type and M4243 viruses in 293T cells transfected with the plasmids. Viral titers were determined by focus-forming assays in Vero cells. (C) Complementation of M4243 in 293T cells transfected with the plasmids at 24 h postinoculation. Viral RNA levels were determined by the negative-strand-specific real-time RT-PCR. Means from three experiments are indicated.
FIG. 8.
FIG. 8.
Virulence of wild-type and M4243 viruses for ICR mice. (A) The infectious titers of wild-type and M4243 viruses in the brains of mice after inoculation with 100 FFU of the viruses intracerebrally (ic) or 105 FFU intraperitoneally (ip). Open circles and closed squares indicate wild-type and M4243 viruses, respectively. The detection limit is 102 FFU/gram of brain. Means of titers from four mice are indicated. (B) Percentages of surviving mice (10 to 11 mice per group) inoculated with 105 FFU of wild-type or M4243 viruses by an intraperitoneal route. Open circles, closed squares, and gray triangles indicate mice infected with the wild-type virus, infected with the M4243 virus, or mock infected, respectively.
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