Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Mar 20:8:128.
doi: 10.1186/1743-422X-8-128.

Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity

Yi-Ping Wu  1 , Chung-Ming Chang, Chun-Yu Hung, Meng-Chieh Tsai, Scott C Schuyler, Robert Yung-Liang Wang
Affiliations

Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity

Yi-Ping Wu et al. Virol J. .

Erratum in

  • Virol J. 2011;8:338

Abstract

The serum-free medium from Japanese encephalitis virus (JEV) infected Baby Hamster Kidney-21 (BHK-21) cell cultures was analyzed by liquid chromatography tandem mass spectrometry (LC-MS) to identify host proteins that were secreted upon viral infection. Five proteins were identified, including the molecular chaperones Hsp90, GRP78, and Hsp70. The functional role of GRP78 in the JEV life cycle was then investigated. Co-migration of GRP78 with JEV particles in sucrose density gradients was observed and co-localization of viral E protein with GRP78 was detected by immunofluorescence analysis in vivo. Knockdown of GRP78 expression by siRNA did not effect viral RNA replication, but did impair mature viral production. Mature viruses that do not co-fractionate with GPR78 displayed a significant decrease in viral infectivity. Our results support the hypothesis that JEV co-opts host cell GPR78 for use in viral maturation and in subsequent cellular infections.

PubMed Disclaimer

Figures

Figure 1
Figure 1
An assay for the collection of secreted proteins from JEV-infected BHK-21 cells. A) A flowchart outlining the protocol for the identification of JE virus replication from intracellular lysates and secreted proteins from the JEV-infected cells. B) Effect of serum deprivation on JEV replication. Cells were incubated with medium supplemented with 2% FBS or serum-free medium for 24 hours and viral proteins, NS1 and NS5, were assessed by Western blot analysis using anti-NS5/anti-NS1 polyclonal antibodies. Extracts from mock-infected cells serve as a negative control. Two independent replicates are shown for the serum-free and 2% serum conditions. C) Effect of serum deprivation on cell viability. β-actin was not detected in the secreted medium of mock- and JEV-infected BHK-21 cells. Three independent replicates are shown for the mock- and JEV-infected conditions.
Figure 2
Figure 2
Proteins identified by LC-MS in the secretion medium of JEV-infected BHK-21 cells. Cell extracts were collected at three days after mock- and JEV-infection and subjected to one dimension SDS-PAGE analysis. A total amount of 10 μg of secreted proteins from mock- and JEV-infected BHK-21 cells was loaded per lane. The gel was stained with Silver nitrate. The identified proteins are shown as Table 1.
Figure 3
Figure 3
GRP78 is released into the media upon JEV infection and partially co-fractionates with the JE virion. A) Verification of GRP78 in the secretome from JEV-induced BHK-21 cells. Cell lysates or secretion medium were collected 3 days post-infection followed by SDS-PAGE for protein separation. The GRP78 was detected by anti-GRP78 specific antibody. Two independent replicates are shown for the mock- and JEV-infected conditions. B) Sucrose density gradient fraction of JE virion and GPR78. A volume of 40 μL of sample from each fraction was analyzed on SDS-PAGE followed by the detection of anti-JEV E protein and anti-GRP78 by Western blotting.
Figure 4
Figure 4
The co-localization of GRP78 and JEV E protein in JEV-infected BHK-21 cells. Mock- or JEV-infected BHK-21 cells were harvested at 3 days post-infection and prepared for immunofluorescence analysis stained with antibodies that detect GRP78 (green; b, f, j) and JEV-E protein (red; a, e, i).
Figure 5
Figure 5
Knocking-down the expression of GRP78 does not affect the replication of JE viral RNA. BHK-21 cells were transfected with siRNA against GRP78 or an irrelevant siRNA (scramble siRNA) for 48 hours. The expression level of GRP78 was measured by Western blot analysis using polyclonal antibody specific to GRP78. Transfected cells were then infected with JEV at an MOI of 1. At 24 hour post-infection, the cell lysates were collected to measure the JEV replication using antibodies specific to NS1 and/or NS5. Two independent replicates are shown for the scramble and GRP78 siRNA conditions
Figure 6
Figure 6
Knocking-down the expression of GRP78 by siRNA decreases the yield of infectious JE virus production. BHK-21 cells were transfected with siRNA against GRP78 or an irrelevant siRNA (negative control). The down-regulation of GRP78 was measured by Western blot analysis using antibody specific to GRP78 as shown in Figure 5. A) Transfected cell lysates were then infected with JEV at an MOI of 1. At 24 hours post-infection, the supernatants were collected to measure the amount of JE viral RNA production by RT real-time PCR as described in Material and Methods. The virus yield is expressed as a percentage of the yield obtained from cells transfected with irrelevant siRNA. B) Plaque formation by JE virus-particle collected from JEV-infected scramble siRNA treated cells or cells treated with siRNA against GRP78. C) Quantitative measurement of viral progeny produced from JEV-infected scramble siRNA treated cells or cells treated with siRNA against GRP78. The virus titer is defined as plaque-forming unit (PFU) per mL. Results are derived from three independent experiments.
Figure 7
Figure 7
Decrease in JEV infectivity in the absence of co-migrating GRP78. A) Viral infectivity using plaque assay of JE virion-fractions associated without or with GRP78 was determined. B) Quantitative measurement of viral progeny produced from E+GRP78 and E only fractionates. The plaque assay results shown here are representatives of three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Calisher CH, Gould EA. Taxonomy of the virus family Flaviviridae. Adv Virus Res. 2003;59:1–19. full_text. - PubMed
    1. Solomon T. Flavivirus encephalitis. N Engl J Med. 2004;351(4):370–378. doi: 10.1056/NEJMra030476. - DOI - PubMed
    1. Chambers TJ, Hahn CS, Galler R, Rice CM. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990;44:649–688. doi: 10.1146/annurev.mi.44.100190.003245. - DOI - PubMed
    1. Markoff L. 5'- and 3'-noncoding regions in flavivirus RNA. Adv Virus Res. 2003;59:177–228. full_text. - PMC - PubMed
    1. Heinz FX, Allison SL. Flavivirus structure and membrane fusion. Adv Virus Res. 2003;59:63–97. full_text. - PubMed

Publication types

Substances

-