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.

Inhibition of transient receptor potential vanilloid 1 (TRPV1) channel regulates chikungunya virus infection in macrophages.

Chikungunya virus (CHIKV), a virus that induces pathogenic inflammatory host immune responses, is re-emerging worldwide, and there are currently no established antiviral control measures. Transient receptor potential vanilloid 1 (TRPV1), a non-selective Ca2+-permeable ion channel, has been found to regulate various host inflammatory responses including several viral infections. Immune responses to CHIKV infection in host macrophages have been reported recently. However, the possible involvement of TRPV1 during CHIKV infection in host macrophages has not been studied. Here, we investigated the possible role of TRPV1 in CHIKV infection of the macrophage cell line RAW 264.7. It was found that CHIKV infection upregulates TRPV1 expression in macrophages. To confirm this observation, the TRPV1-specific modulators 5'-iodoresiniferatoxin (5'-IRTX, a TRPV1 antagonist) and resiniferatoxin (RTX, a TRPV1 agonist) were used. Our results indicated that TRPV1 inhibition leads to a reduction in CHIKV infection, whereas TRPV1 activation significantly enhances CHIKV infection. Using a plaque assay and a time-of-addition assay, it was observed that functional modulation of TRPV1 affects the early stages of the viral lifecycle in RAW 264.7 cells. Moreover, CHIKV infection was found to induce of pNF-κB (p65) expression and nuclear localization. However, both activation and inhibition of TRPV1 were found to enhance the expression and nuclear localization of pNF-κB (p65) and production of pro-inflammatory TNF and IL-6 during CHIKV infection. In addition, it was demonstrated by Ca2+ imaging that TRPV1 regulates Ca2+ influx during CHIKV infection. Hence, the current findings highlight a potentially important regulatory role of TRPV1 during CHIKV infection in macrophages. This study might also have broad implications in the context of other viral infections as well.

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.

Chikungunya virus nsP1 interacts directly with nsP2 and modulates its ATPase activity.

Chikungunya virus (CHIKV) is a mosquito-borne virus, which has created an alarming threat in the world due to unavailability of vaccine and antiviral compounds. The CHIKV nsP2 contains ATPase, RTPase, helicase and protease activities, whereas, nsP1 is a viral capping enzyme. In alphaviruses, the four non-structural proteins form the replication complex in the cytoplasm and this study characterizes the interaction between CHIKV nsP1 and nsP2. It was observed that, both the proteins co-localize in the cytoplasm and interact in the CHIKV infected cells by confocal microscopy and immunoprecipitation assay. Further, it was demonstrated through mutational analysis that, the amino acids 1-95 of nsP2 and 170-288 of nsP1 are responsible for their direct interaction. Additionally, it was noticed that, the ATPase activity of nsP2 is enhanced in the presence of nsP1, indicating the functional significance of this interaction. In silico analysis showed close (≤1.7 Å) polar interaction (hydrogen bond) between Glu4, Arg7, 96, 225 of nsP2 with Lys256, 206, Val367 and Phe312 of nsP1 respectively. Hence, this investigation provides molecular characterization of CHIKV nsP1-nsP2 interaction which might be a useful target for rational designing of antiviral drugs.

Regulation of Viral Replication, Apoptosis and Pro-Inflammatory Responses by 17-AAG during Chikungunya Virus Infection in Macrophages.

Chikungunya virus (CHIKV) infection has re-emerged as a major public health concern due to its recent worldwide epidemics and lack of control measures. Although CHIKV is known to infect macrophages, regulation of CHIKV replication, apoptosis and immune responses towards macrophages are not well understood. Accordingly, the Raw264.7 cells, a mouse macrophage cell line, were infected with CHIKV and viral replication as well as new viral progeny release was assessed by flow cytometry and plaque assay, respectively. Moreover, host immune modulation and apoptosis were studied through flow cytometry, Western blot and ELISA. Our current findings suggest that expression of CHIKV proteins were maximum at 8 hpi and the release of new viral progenies were remarkably increased around 12 hpi. The induction of Annexin V binding, cleaved caspase-3, cleaved caspase-9 and cleaved caspase-8 in CHIKV infected macrophages suggests activation of apoptosis through both intrinsic and extrinsic pathways. The pro-inflammatory mediators (TNF and IL-6) MHC-I/II and B7.2 (CD86) were also up-regulated during infection over time. Further, 17-AAG, a potential HSP90 inhibitor, was found to regulate CHIKV infection, apoptosis and pro-inflammatory cytokine/chemokine productions of host macrophages significantly. Hence, the present findings might bring new insight into the therapeutic implication in CHIKV disease biology.

Gene expression profile of rat prostate during pubertal growth and maturation.

Temporal gene expression profiling can provide valuable insight into mechanisms of differentiation and may be helpful in laying a foundation for characterization of the molecular aspects of development. Prostate development begins in fetal life and is complete at sexual maturity, and androgen stimulation is both necessary and sufficient for development and maturity of the prostate. In this study, we investigated gene expression profiles of rat prostate at 3 different developmental stages (2 weeks, 3.5 weeks, and 8 weeks), when serum testosterone levels are low, intermediate, and high. Through this analysis, we attempted to narrow down genes whose expression is affected by androgen increase during pubertal growth and maturation of the prostate.

Overexpression of hepatocyte nuclear factor-3alpha induces apoptosis through the upregulation and accumulation of cytoplasmic p53 in prostate cancer cells.

BACKGROUND: Hepatocyte nuclear factor-3alpha (HNF-3alpha) has been known to act as a repressor in the pathogenesis of many cancers. Herein, we investigated the effect of HNF-3alpha overexpression in prostate cancer cells. METHODS: HNF-3alpha was overexpressed in prostate cancer cells using an adenovirus recombinant expressing wild-type HNF-3alpha. The apoptosis of prostate cancer cells was determined by TUNEL, FACS, and caspase activity analyses. RESULTS: Adenovirus-mediated overexpression of HNF-3alpha caused cell death in prostate cancer cells as assessed by changes in cellular and nuclear morphology, TUNEL analysis, and caspase activations. Furthermore, FACS analysis showed an increased sub-G1 phase of cell cycle as well as the G2/M phase with a corresponding decrease in S phases. HNF-3alpha overexpression caused the upregulation of p53 protein and its accumulation, together with HNF-3alpha, in the cytoplasm. It also causes Bax protein to localize to the mitochondria-enriched fraction. These findings suggest that multiple apoptotic pathways seem to be involved in the HNF-3alpha-induced cell death: pathways involving the accumulation of p53 protein in the cytoplasm and subsequent cytochrome c release, and other pathways involving death receptor signaling and caspase-8 activation. CONCLUSIONS: The results of the current study suggest a novel function of HNF-3alpha as a killer of malignant prostate cancer cells, which reveals HNF-3alpha as a promising therapeutic molecule for prostate cancers.

Hepatocyte nuclear factor-3 alpha (HNF-3alpha) negatively regulates androgen receptor transactivation in prostate cancer cells.

The androgen receptor (AR) is involved in the development and progression of prostate cancers. However, the mechanisms by which this occurs remain incompletely understood. In previous reports, hepatocyte nuclear factor-3alpha (HNF-3alpha) has been shown to be expressed in the epithelia of the prostate gland, and has been determined to regulate the transcription of prostate-specific genes. In this study, we report that HNF-3alpha functions as a novel corepressor of AR in prostatic cells. HNF-3alpha represses AR transactivation on target promoters containing the androgen response element (ARE) in a dose-dependent manner. HNF-3alpha interacts physically with AR, and negatively regulates AR transactivation via competition with AR coactivators, including GRIP1. Furthermore, HNF-3alpha overexpression reduces the androgen-induced expression of prostate-specific antigen (PSA) in LNCaP cells. Taken together, our findings indicate that HNF-3alpha is a novel corepressor of AR, and predict its effects on the proliferation of prostate cancer cells.

The CCAAT enhancer-binding protein-alpha negatively regulates the transactivation of androgen receptor in prostate cancer cells.

The basic leucine zipper transcription factor, CCAAT enhancer-binding protein-alpha (C/EBPalpha), negatively regulates cell proliferation and induces terminal differentiation of various cell types. C/EBPalpha is expressed in the prostate, but its potential role in the tissue is unknown. Herein, we show that C/EBPalpha is highly expressed at the stage of growth arrest during prostate development. Furthermore, overexpression of C/EBPalpha decreases the rate of DNA synthesis in LNCaP prostate cancer cells. Investigation of the potential cross-talk between C/EBPalpha and androgen receptor (AR) that is responsible for androgen-dependent prostate proliferation demonstrates that androgen-dependent transactivation of AR is strongly repressed by C/EBPalpha. C/EBPalpha directly binds AR in vitro and forms a complex with AR in vivo. C/EBPalpha neither prevents the nuclear translocation of AR nor disrupts the N/C-terminal interaction of AR, which are both necessary for its proper transactivation activity upon ligand binding. To modulate AR transactivation, however, C/EBPalpha does compete with AR coactivators for AR binding. Additionally, C/EBPalpha is recruited onto AR-target promoters with AR and is further able to inhibit the expression of endogenous prostate-specific antigen in prostate cancer cells. Our results suggest C/EBPalpha as a potent AR corepressor and provide insight into the role of C/EBPalpha in prostate development and cancer.

Androgen receptor corepressor-19 kDa (ARR19), a leucine-rich protein that represses the transcriptional activity of androgen receptor through recruitment of histone deacetylase.

Androgen receptor (AR) that mediates androgen action is a crucial factor in male reproductive functions. Here, we report a novel AR corepressor ARR19 (androgen receptor corepressor-19 kDa), which has been isolated as a putative androgen-induced gene from murine testis. ARR19 encoding a leucine-rich protein is expressed only in male reproductive organs such as testis and prostate. ARR19 expression in the testis is developmentally regulated. Functional analysis conducted by the transient transfection of mammalian cells shows that ARR19 represses AR transactivation in a dose-dependent manner. Furthermore, yeast two-hybrid and glutathione S-transferase pull-down analyses reveal that ARR19 directly associates with AR through the N-terminal and leucine zipper-containing regions of ARR19 and the DNA binding-hinge domain of AR. Interestingly, ARR19 localized in the cytoplasmic compartment cotranslocates into the nucleus with AR upon androgen exposure. The ARR19 repression of AR transactivation is through the recruitment of histone deacetylase 4 (HDAC4) by ARR19. Overexpression of HDAC4 further inhibits the ARR19-repressed AR transactivation. In addition, ARR19 directly interacts with HDAC4 in vitro. Furthermore, DNA-protein complex immunoprecipitation assays reveal that HDAC4 is recruited to an androgen-regulated promoter through ARR19. Taken together, the results suggest that ARR19 may act as an AR corepressor in vivo and play an important role in male reproductive functions.