key: cord-0336778-mltb8jou
authors: Sui, Liyan; Zhao, Yinghua; Wang, Wenfang; Chi, Hongmiao; Tian, Tian; Wu, Ping; Zhang, Jinlong; Zhao, Yicheng; Wei, Zheng-Kai; Hou, Zhijun; Zhou, Guoqiang; Wang, Guoqing; Wang, Zedong; Liu, Quan
title: Flavivirus prM interacts with MDA5 and MAVS to inhibit RLR antiviral signaling
date: 2022-04-25
journal: bioRxiv
DOI: 10.1101/2022.04.25.489335
sha: befaf04c0956d163fe4e8e3d268cf97f2cc857f1
doc_id: 336778
cord_uid: mltb8jou

Vector-borne flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKA), West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), pose a growing threat to public health worldwide, and have evolved complex mechanisms to overcome host antiviral innate immunity. However, the underlying mechanisms of flavivirus structural proteins to evade host immune response remain elusive. Here we show that TBEV structural proteins, including pre-membrane (prM), envelope, and capsid proteins, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, thereby impeded the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral signaling. ZIKA and WNV prM was also demonstrated to interact with both MDA5 and MAVS, while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. Overexpression of TBEV and ZIKA prM significantly promoted the replication of Sendai virus. Our findings reveal the immune evasion mechanisms of flavivirus prM, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention and vaccine development. Author Summary All flaviviruses must overcome the antiviral innate immunity to infect vertebrate host. The non-structural proteins of flaviviruses are mainly responsible for viral replication and host innate immune escape, and the structural proteins are involved in the virus assembly, which are potential targets for prevention and treatment of flaviviral infections. Whether flavivirus structural proteins participate in host innate immune escape remains to be determined. Here, we found that tick-borne encephalitis virus structural proteins precursor membrane (prM), capsid, and envelope proteins can antagonize type I interferon production, in which prM interacts with both MDA5 and MAVS to inhibit RLR antiviral signaling. Additionally, Zika virus and West Nile virus prMs also interact with both MDA5 and MAVS, while dengue virus serotype 2 and yellow fever virus prMs associate only with MDA5 or MAVS to suppress type I interferon production. In contrast, Japanese encephalitis virus prM cannot antagonize type I interferon production. Our findings reveal that flavivirus prM inhibits type I interferon production via interacting with MDA5 and/or MAVS in a species-dependent manner, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention, and vaccine development.

we show that TBEV structural proteins, including pre-membrane (prM), envelope, and capsid 23 proteins, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted 24 with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, 25 thereby impeded the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral 26 signaling. ZIKA and WNV prM was also demonstrated to interact with both MDA5 and MAVS, 27 while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to 28 suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. 29

Overexpression of TBEV and ZIKA prM significantly promoted the replication of Sendai virus. C and E could function as interferon antagonists (Fig. 1a) . Although the C protein exhibited higher 117 inhibitory effect than prM and E protein, its cytotoxicity was much higher than that of the prM 118 protein ( Supplementary Fig. S1d) . 119

The inhibitory effect of TBEV prM was further confirmed by luciferase reporter assay. PrM 120 protein was shown to significantly inhibit the promoter activity of IFNβ and ISRE (IFN-sensitive 121 responsive element) induced by poly (I:C) (Fig. 1b, c) , and overexpression of prM suppressed the 122 activity of NF-κB promoter induced by RIG-I-N (the N-terminal CARD domain of RIG-I) (Fig.  123 1d). TBEV prM also significantly inhibited the mRNA levels of IFNB1, ISG56, and CXCL10 (Fig.  124 1e). 125

The activation of IRF3, including its phosphorylation, dimerization and nuclear translocation 126 are important for IFN-I production [28] . We thus examined if TBEV prM suppresses the activation 127 of IRF3. Compared with empty vector, TBEV prM overexpression slightly reduced the 128 phosphorylation of IRF3 and TBK1 induced by poly (I:C) (Fig. 1f) , while the dimerization of 129 IRF3 was significantly reduced in the prM transfection group (Fig. 1g) . Moreover, the expression 130 of IRF3 in nuclei activated by poly (I:C) was decreased in the prM expression group (Fig. 1h) . The RLRs members, such as RIG-I and MDA5, are important sensors of cytosolic viral RNA, which 157 play a critical role in IFN-I production ( Fig. 2a ) [8] . We thus examined the effect of TBEV prM on 158 RLR-mediated IFN-I production, and found that co-expression of TBEV prM suppressed IFNβ 

With the exception of TBEV, several flaviviruses, including DENV2, JEV, YFV, WNV and ZIKA, 13 also pose severe threats to human health, whose prMs share 14-40% amino acid similarities with 240 that of TBEV ( Fig. 5a and Supplementary Table S1). ZIKA prM has shown to suppress IFN-I 241 production, whereas DENV2 prM has no significant effect on interferon production [23, 24] . 242

Next, we investigated if these flavivirus prMs antagonize host innate immunity as the same way as 243 TBEV prM. interaction with RIG-I (Fig. 6a) , while prMs of DENV2, WNV, ZIKA, and TBEV interacted with 270 MDA5 (Fig. 6d) . YFV, WNV, ZIKA, and TBEV prMs could bind to MAVS, with a little stronger 271 interaction for ZIKA and TBEV prMs as comparison with YFV and WNV (Fig. 6c) . In contrast, 272 none of the flavivirus prMs interacted with TBK1 ( Supplementary Fig. S5) . SeV, higher in 1 μg than in 0.5 μg prM transfection group (Fig. 7a) . Flow cytometry analysis showed 296 that along with the increasing dose of prM transfected, the percentage of SeV-positive cells was 297 gradually elevated, significantly higher in the 1.0 μg prM transfection group as comparison with the 298 control group (Supplementary Fig. S6 ). Immunoblot analysis also showed a significantly higher 299

SeV protein levels in prM transfection group compared with the empty vector and control group 300 (Fig. 7b) . The TM and mature M domain that interacted with MDA5 and MAVS obviously promoted 301

SeV replication (Fig. 7c) . Given the interferon antagonizing activity of the flaviviruses, we further 302 detected the effect of prMs from DENV2, JEV and ZIKA on SeV replication. Similar to TBEV, 303 ZIKA prM significantly promoted replication of SeV, while DENV2 and JEV prM proteins showed 304 no significant effect (Fig. 7d) . Consistently, flow cytometry analysis revealed that the full length 305 and the 130-164 aa truncation of TBEV and ZIKA prMs significantly enhanced the percentage of 306

SeV-positive cells, while the SeV-positive cells in DENV2 prM transfection group was a little bit 307 higher than the empty vector group, and JEV prM did not affect SeV production (Fig. 7e) . Taken 308 together, TBEV and ZIKA prMs can facilitate SeV replication. were conducted in A and the data were showed in column graph. *p < 0.05, **p < 0.01. 493 494

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