Delayed IFN response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells

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1 Journal of General Virology (),, DOI./vir.. Short Communication Delayed IFN response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells Yuki Takamatsu, Leo Uchida and Kouichi Morita Correspondence Kouichi Morita Received March Accepted 7 April Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important causes of human encephalitis cases, which result in a high mortality ratio and neurological sequelae after recovery. Understanding the mechanism of neuropathogenicity in these viral infections is important for the development of specific antiviral therapy. Here, we focused on human-derived neuronal and glial cells to understand the cellular responses against WNV and JEV infection. It was demonstrated that early IFN-b induction regulated virus replication in glioblastoma TG cells, whereas delayed IFN-b induction resulted in efficient virus replication in neuroblastoma cells. Moreover, the concealing of viral dsrna in the intracellular membrane resulted in the delayed IFN response in cells. These results, which showed different IFN responses between human neuronal and glial cells after WNV or JEV infection, are expected to contribute to our understanding of the molecular mechanisms for neuropathology in these viral infections. West Nile virus (WNV) and Japanese encephalitis virus (JEV) are mosquito-borne viruses that belong to the genus Flavivirus, family Flaviviridae (Gubler et al., 7; Sips et al., ). Most infections, due to either virus, are asymptomatic or cause febrile illness in humans, and may lead to encephalitis resulting in a high mortality ratio and neurological sequelae after recovery (Solomon, ). Specific antiviral therapy has not yet been developed (Solomon, ). There have been few reports focused on WNV and JEV infection in human cells derived from brain (Kleinschmidt et al., 7; Kumar et al., ) and it has not yet been revealed how these viruses spread in these cells. The mechanism of neuropathogenicity in WNV and JEV infection is an important research interest for the development of specific antiviral therapy. WNV and JEV can multiply in several cultured cell types, which have a cytopathic effect (CPE) upon infection (Gubler et al., 7; Kleinschmidt et al., 7; Kumar et al., ; Parquet et al., ; Stim & Henderson, ). To understand the cellular responses against WNV and JEV infection, we used two kinds of cells, which exhibit different IFN responses and virus replication rates after WNV and JEV infection. The IFN response is an important defence against the early phase of viral infections (Randall & Goodbourn, ). Type I IFN induction is triggered by viral components One supplementary figure and one supplementary table are available with the online Supplementary Material. called pathogen-associated molecular patterns (PAMPs) such as virus-derived double-stranded RNA (dsrna). The endoplasmic reticulum (ER) provides the membrane platform for the formation of the flavivirus replication complex, which houses the non-structural (NS) proteins and the accumulating viral dsrnas during viral genome synthesis (Gillespie et al., ; Westaway et al., 7). The dsrna is recognized by pattern recognition receptors (PRRs), namely retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein (MDA), and induces the IFN response (Loo & Gale, ; Quicke & Suthar, ). The induction of IFN-a/b is critical for controlling WNV and JEV replication during the course of infection (Lin et al., ; Quicke & Suthar, ). It was initially reported in tick-borne encephalitis virus (TBEV) infection that the dsrna is enclosed in intracellular membrane vesicles and, as a consequence, escapes from the host immune system (Överby et al., ). It was reported previously in JEV-infected porcine cells that viral dsrna is concealed in an intracellular membrane resulting in delayed cytosolic exposure (Espada-Murao & Morita, ). The mechanism of concealing dsrna was also reported in dengue virus (DENV)-infected HeLa cells, which resulted in the evasion of IFN response (Uchida et al., ). These findings clearly suggest that the concealing of dsrna plays a crucial role in virus replication, leading to escape from the host immune system in flavivirus infection. This study aimed to reveal the molecular mechanisms for WNV and JEV dissemination and replication in human neuronal and glial cells. G The Authors Printed in Great Britain IP:... On: Fri, Aug ::

2 IFN response of neuronal and glial cells to WNV and JEV (a) WNV Log p.f.u. ml NY... Log p.f.u. ml Egypt JaOArS JaNAr JEV Log p.f.u. ml. Log p.f.u. ml. TG P<. (b) NY Egypt IFN-β Log.. <... IFN-β Log..... IFN-β Log JaOArS....7 IFN-β Log JaNAr... TG Fig.. Virus replication and IFN-b induction in cells and TG cells. (a) Each virus (WNV; NY, Egyot, JEV; JaOArS, JaNAr) was inoculated at an m.o.i. of onto cells and TG cells. The cell supernatants were harvested at,,,,, and h post-inoculation. The results of p.f.u. titration are expressed as the mean of three independent experiments; the error bars indicate SD. A group of samples was assessed by Student s t-tests or Welch tests. A P value of less than. was considered statistically significant. Double asterisks (P,.) and a number indicate the P value. (b) Each virus (WNV; NY and Egyot, JEV; JaOArS and JaNAr) was inoculated on a monolayer of SK- N-SH cells and TG cells in -well plates at an m.o.i. of. The number of copies of IFN-b mrna and glyceraldehyde - phosphate dehydrogenase (GAPDH) mrna was calculated by absolute quantification based on in vitro-transcribed viral RNA, and GAPDH standards. The results for IFN-b mrna were normalized to GAPDH and expressed as the fold increase over non-infected cells. The results are expressed as the mean of three independent experiments, and the error bars indicate SD. A group of samples was assessed by Student s t-tests or Welch tests. A P value of less than. was considered statistically significant. A number indicates the P value. IP:... On: Fri, Aug ::

3 Y. Takamatsu, L. Uchida and K. Morita (a) TG Control A B WNV JEV WNV JEV (b) TG Log p.f.u. ml 7 WNV (m.o.i. ) 7 Log p.f.u. ml WNV (m.o.i. ) Log p.f.u. ml 7 JEV (m.o.i. ) P<. Control Units Units (c) TG Non-infection WNV- h WNV- h JEV- h JEV- h Non-infection WNV- h WNV- h JEV- h JEV- h WNV (m.o.i. ) Log p.f.u. ml 7 RIG-l kda β-actin kda Fig.. IFN response and cytosolic PRRs expression. (a) WNV (NY) or JEV (JaOArS) infection was performed on a monolayer of cells in -well plates with or without anti-ifn cocktail treatment. The cells were treated with anti-ifn cocktail h before viral inoculation. Culture medium was used for control cells. At days post-inoculation, the size of foci was compared by a focus forming assay. (A) neutralization units anti-human IFN-b ml and mg anti-human IFN-a/bR ml. (B) neutralization units ml anti-human IFN-b and mg anti-human IFN-a/bR ml. (b) WNV (NY) or JEV (JaOArS) infection was performed on a monolayer of cells in -well plates at an m.o.i. of, with or without immediate IFN-b treatment ( or units per well). Culture medium was used for control cells. At and h post-inoculation, viral titres were measured by p.f.u. titration. The results are expressed as the mean of three independent experiments, and the error bars indicate SD. A group of samples was assessed by Student s t-tests or Welch tests. A P value of less than. was indicated as an asterisk. (c) WNV (NY) or JEV (JaOArS) infection was performed on a monolayer of cells in - well plates at an m.o.i. of. At and h post-inoculation, the infected cells were harvested and the target proteins were detected by immunoblotting. b-actin was used as an internal control. All the samples were derived from the same experiment and blotting was processed in parallel. Journal of General Virology IP:... On: Fri, Aug ::

4 IFN response of neuronal and glial cells to WNV and JEV (a) Digitonin TG NP- Digitonin NP- (b) dsrna exposed ratio (%) TG P<. (c) dsrna exposed ratio (%) TG P<. Fig.. Delayed cytosolic exposure of dsrna in cells. WNV (NY) infection was performed on a monolayer of cells in eight-well chamber slides at an m.o.i. of. (a) At the indicated time points, the infected cells were fixed and permeabilized with % NP- or. mm digitonin. The viral dsrna was visualized using an immunofluorescence assay. Nuclear staining was achieved using DAPI. (b, c) The exposed dsrna ratio in WNV (NY)-infected (b) or JEV (JaOArS)-infected (c) cells and TG cells. The ratio, expressed as a percentage, was determined by dividing the number of digitonin-treated cells positive for dsrna by the number of NP--treated cells positive for dsrna. The images showed comparable numbers of total cells from both NP-- and digitonin-treated cells. The percentage of cells was calculated in three different fields. The error bars indicate SEM. A group of samples was assessed by a Mann Whitney U test. A P value of less than. was considered statistically significant, and is indicated as an asterisk. IP:... 7 On: Fri, Aug ::

5 Y. Takamatsu, L. Uchida and K. Morita Human neuroblastoma cells (HTB-; ATCC), and human glioblastoma TG cells (CRL-; ATCC) were used for virus infection. The role of glial cells in WNV and JEV infection is unknown, and this is the first attempt to compare WNV and JEV infection in neuronal cells and glial cells. WNV strains NY and Egypt and JEV strains JaOArS and JaNAr were used in this study. The viruses were propagated in African green monkey kidney cells (Vero cells) to generate working stocks. At 7 h post-inoculation, the culture supernatants were collected and stored in aliquots at C. WNV or JEV infection was performed on a monolayer of cells, in -well plates, at an m.o.i. of, and supernatants were harvested at the indicated time points. Viral titres were determined by plaque-forming assays on BHK cells and expressed as p.f.u. ml (Hayasaka et al., ; Takamatsu et al., ). Clear CPE (rounding of cells, detachment from the monolayer and cell shrinkage) was observed in WNV- or JEV-infected cells from days postinoculation. On the other hand, no clear morphology changes were observed in WNV- or JEV-infected TG cells up to days post-inoculation (data not shown). SK- N-SH cells showed higher viral titres than TG cells after infection with WNV or JEV. A slower viral growth and a lower peak of viral titre were observed in TG cells (Fig. a). These experiments suggested that cells are permissive for virus replication, whereas TG cells have some mechanisms to regulate virus propagation. Next, we focused on cellular innate immune response against viral infection. Real-time quantitative reverse transcription PCR for IFN-b was performed as described previously (Takamatsu et al., ). The primer information is indicated in Table S, available in the online Supplementary Material. Early IFN-b upregulation was observed both in WNV- and JEV-infected TG cells from h postinoculation (Fig. b). The timing of increased viral RNA levels and virus production was similar in cells and TG cells (Fig. S). Interestingly, delayed IFN-b induction was shown in cells, although sufficient virus replication was observed from an early time of infection. On the other hand, immediate IFN-b induction was shown in the absence of sufficient virus replication in TG cells. There was no significant difference in the basal levels of IFN-b (absolute amount) between SK-N- SH and TG cells prior to infection (data not shown). To clarify the role of type I IFN in virus dissemination and growth, it was blocked using an anti-ifn antibody cocktail. The cells were treated with a combination of an antihuman IFN-b (PBL Interferon Source) and an antihuman IFN-a/bR (PBL Interferon Source) at different concentrations h before the viral inoculation (Uchida et al., ). A focus formation assay was performed on cells and TG cells as described previously (Espada-Murao & Morita, ). Foci of infected cells were larger in WNV- or JEV-infected and TG cells treated with anti-ifn cocktail compared with the non-treated group (Fig. a). These results indicate that IFN response restricts virus replication change to in both and TG cells. To elucidate the role of IFN-b on virus replication, an immediate IFN-b treatment with or units per well was performed (Espada- Murao & Morita, ). The results showed that early IFN-b treatment significantly reduced viral titres in both cells and TG cells (Fig. b). It is suggested that the delayed IFN-b induction in cells impaired the IFN response during early infection, thereby enhancing WNV and JEV replication. The cytosolic PRRs are reported to regulate IFN-a/b expression in flavivirus infection (Kato et al., ; Loo & Gale, ). To compare protein expression in WNV- and JEV-infected cells, cellular extracts were subjected to immunoblotting for RIG-I, MDA and for b-actin as an internal control (Takamatsu et al., ; Uchida et al., ). RIG-I expression was detectable from h post-inoculation in both and TG cells (Fig. c), whereas MDA expression was not detectable in the two cells (data not shown). It indicates that the RIG-I, but not MDA-, is required for IFN-b induction in JEV infection (Kato et al., ), and this is in agreement with a previous report (Uchida et al., ). The timing of the dsrna exposure in the cytosol has been reported to be important for inducing an IFN response in TBEV-, DENV- and JEV-infected cells (Espada-Murao & Morita, ; Överby et al., ; Uchida et al., ). Two permeabilization methods were applied to differentiate the localization of the dsrna, either exposed in cytoplasm or concealed in intracellular membrane. Nonidet P- (NP-) treatment permeabilizes all cellular membrane structures, including the plasma membrane and ER; digitonin permeabilizes only the plasma membrane (Uchida et al., ). WNV or JEV infection was performed on a monolayer of cells in eight-well chamber slides (Nunc) at an m.o.i. of. At indicated time points, the cells were subjected to immunostaining as described previously (Espada- Murao & Morita, ; Uchida et al., ). The mouse IgGa K mab (English & Scientific Consulting) was used to visualize viral dsrna. The images were captured using a LSM 7 confocal laser scanning microscope (Carl Zeiss). The cell number in a field was counted by ImageJ software (Schneider et al., ). Interestingly, the dsrna was predominantly concealed in intracellular membrane structures in cells after WNV infection (Fig. a). The ratio of exposed dsrna in cytoplasm was lower (approx. %) in WNV-infected cells, whereas that in TG cells was higher (approx. %) during the course of infection (Fig. b). A similar finding of concealed dsrna was noted in JEV-infected cells, and the ratio of exposed dsrna was also higher in TG cells than that in cells (Fig. c). The results suggested that the delayed cytosolic exposure of dsrna was correlated with the delayed IFN-b induction in SK- N-SH cells. In our previous report, the dsrna of DENV was concealed in intracellular membranes but JEV was exposed in the cytoplasm of human cervical-derived HeLa cells during early infection (Uchida et al., ). IP:... Journal of General Virology On: Fri, Aug ::

6 IFN response of neuronal and glial cells to WNV and JEV A similar finding was observed in WNV-infected HeLa cells (data not shown). The specific observation of concealed dsrna in neuronal cells may contribute to the viral pathogenicity in WNV and JEV infection. It is suggested that dsrna leaking from the small pore of replication vesicles during the later phase of infection is recognized by cytosolic PRRs in TBEV infection (Överby & Weber, ). It is possible that the difference in formation of vesicle packets, where dsrna is likely to be concealed (Gillespie et al., ), is related to the difference in the levels of dsrna exposure in the cytoplasm between neuronal and glial cells. However, the mechanism of triggering the cytosolic dsrna exposure has not yet been identified. Therefore, we need further investigations to reveal how WNV and JEV utilize the mechanism of concealing viral dsrna in association with host cellular factors. In conclusion, this is the first report to indicate the mechanisms of concealing dsrna in WNV- and JEV-infected human neuronal cells. Early IFN-b induction regulates virus replication in glioblastoma TG cells, whereas delayed IFN-b induction resulted in efficient virus replication in neuroblastoma cells. These results, which showed a difference in the IFN response between human neuronal and glial cells after WNV or JEV infection, are expected to contribute to our understanding of the molecular mechanisms of neuropathology caused by these viruses. Acknowledgements We would like to thank Dr Corazon C. Buerano and Mr Gianne Eduard L. Ulanday from the Department of Virology, Institute of Tropical Medicine, Nagasaki University, for helping the revision of our manuscript, and all of the members and the recent alumni of the Department of Virology, Institute of Tropical Medicine, Nagasaki University, for their support. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; a Grant-in- Aid for JSPS Fellows (Japan Society for the Promotion of Science) from MEXT, Japan; the Global COE program, MEXT, Japan, the Japan Initiative for Global Network on Infectious Diseases (J- GRID), MEXT, Japan; and a Grant-in-aid for scientific research from the Ministry of Health, Labour, and Welfare, Japan. References Espada-Murao, L. A. & Morita, K. (). Delayed cytosolic exposure of Japanese encephalitis virus double-stranded RNA impedes interferon activation and enhances viral dissemination in porcine cells. J Virol, 7 7. Gillespie, L. K., Hoenen, A., Morgan, G. & Mackenzie, J. M. (). The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex. J Virol, 7. Gubler, D. J., Kuno, G. & Markoff, L. (7). Fields Virology. 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