TLR4 is one of the receptors for Chikungunya virus envelope protein E2 and regulates virus induced pro-inflammatory responses in host macrophages.

Toll like receptor 4 (TLR4), a pathogen-associated molecular pattern (PAMP) receptor, is known to exert inflammation in various cases of microbial infection, cancer and autoimmune disorders. However, any such involvement of TLR4 in Chikungunya virus (CHIKV) infection is yet to be explored. Accordingly, the role of TLR4 was investigated towards CHIKV infection and modulation of host immune responses in the current study using mice macrophage cell line RAW264.7, primary macrophage cells of different origins and in vivo mice model. The findings suggest that TLR4 inhibition using TAK-242 (a specific pharmacological inhibitor) reduces viral copy number as well as reduces the CHIKV-E2 protein level significantly using p38 and JNK-MAPK pathways. Moreover, this led to reduced expression of macrophage activation markers like CD14, CD86, MHC-II and pro-inflammatory cytokines (TNF, IL-6, MCP-1) significantly in both the mouse primary macrophages and RAW264.7 cell line, in vitro. Additionally, TAK-242-directed TLR4 inhibition demonstrated a significant reduction of percent E2-positive cells, viral titre and TNF expression in hPBMC-derived macrophages, in vitro. These observations were further validated in TLR4-knockout (KO) RAW cells. Furthermore, the interaction between CHIKV-E2 and TLR4 was demonstrated by immuno-precipitation studies, in vitro and supported by molecular docking analysis, in silico. TLR4-dependent viral entry was further validated by an anti-TLR4 antibody-mediated blocking experiment. It was noticed that TLR4 is necessary for the early events of viral infection, especially during the attachment and entry stages. Interestingly, it was also observed that TLR4 is not involved in the post-entry stages of CHIKV infection in host macrophages. The administration of TAK-242 decreased CHIKV infection significantly by reducing disease manifestations, improving survivability (around 75%) and reducing inflammation in mice model. Collectively, for the first time, this study reports TLR4 as one of the novel receptors to facilitate the attachment and entry of CHIKV in host macrophages, the TLR4-CHIKV-E2 interactions are essential for efficient viral entry and modulation of infection-induced pro-inflammatory responses in host macrophages, which might have translational implication for designing future therapeutics to regulate the CHIKV infection.

MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway.

Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MAPK activated protein kinase 2 (MK2) and MAPK activated protein kinase 3 (MK3) were selected for further analysis. The substrate spectrum of both enzymes is indistinguishable and covers proteins involved in cytokines production, endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling and transcriptional regulation. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during late CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway. Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of the drug against CHIKV. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as important target for developing effective anti-CHIKV strategies.

P38 and JNK Mitogen-Activated Protein Kinases Interact With Chikungunya Virus Non-structural Protein-2 and Regulate TNF Induction During Viral Infection in Macrophages.

Chikungunya virus (CHIKV), a mosquito-borne Alphavirus, is endemic in different parts of the globe. The host macrophages are identified as the major cellular reservoirs of CHIKV during infection and this virus triggers robust TNF production in the host macrophages, which might be a key mediator of virus induced inflammation. However, the molecular mechanism underneath TNF induction is not understood yet. Accordingly, the Raw264.7 cells, a mouse macrophage cell line, were infected with CHIKV to address the above-mentioned question. It was observed that CHIKV induces both p38 and JNK phosphorylation in macrophages in a time-dependent manner and p-p38 inhibitor, SB203580 is effective in reducing infection even at lower concentration as compared to the p-JNK inhibitor, SP600125. However, inhibition of p-p38 and p-JNK decreased CHIKV induced TNF production in the host macrophages. Moreover, CHIKV induced macrophage derived TNF was found to facilitate TCR driven T cell activation. Additionally, it was noticed that the expressions of key transcription factors involved mainly in antiviral responses (p-IRF3) and TNF production (p-c-jun) were induced significantly in the CHIKV infected macrophages as compared to the corresponding mock cells. Further, it was demonstrated that CHIKV mediated TNF production in the macrophages is dependent on p38 and JNK MAPK pathways linking p-c-jun transcription factor. Interestingly, it was found that CHIKV nsP2 interacts with both p-p38 and p-JNK MAPKs in the macrophages. This observation was supported by the in silico protein-protein docking analysis which illustrates the specific amino acids responsible for the nsP2-MAPKs interactions. A strong polar interaction was predicted between Thr-180 (within the phosphorylation lip) of p38 and Gln-273 of nsP2, whereas, no such polar interaction was predicted for the phosphorylation lip of JNK which indicates the differential roles of p-p38 and p-JNK during CHIKV infection in the host macrophages. In summary, for the first time it has been shown that CHIKV triggers robust TNF production in the host macrophages via both p-p38 and p-JNK/p-c-jun pathways and the interaction of viral protein, nsP2 with these MAPKs during infection. Hence, this information might shed light in rationale-based drug designing strategies toward a possible control measure of CHIKV infection in future.