key: cord-0315762-cmyzdt9e authors: Mamidi, Prabhudutta; Nayak, Tapas Kumar; Kumar, Abhishek; Kumar, Sameer; Chatterjee, Sanchari; De, Saikat; Datey, Ankita; Laha, Eshna; Ray, Amrita; Chattopadhyay, Subhasis; Chattopadhyay, Soma title: MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway date: 2021-05-26 journal: bioRxiv DOI: 10.1101/2021.05.26.445768 sha: 4ef82906afb55ff60f22b9ffa877fae64919fe55 doc_id: 315762 cord_uid: cmyzdt9e 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, MK2 and MK3 were selected for further analysis. 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 (a) inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during the late phase of 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 that is responsible for releasing the virus from the infected cells. 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 this drug against CHIKV. Additionally, CMPD1 also inhibited HSV1 and SARS CoV2-19 infection in vitro. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as key targets for developing effective anti-CHIKV strategies in future. Author summary Chikungunya virus has been a dreaded disease from the first time it occurred in 1952 Tanzania. Since then it has been affecting the different parts of the world at different time periods in large scale. It is typically transmitted to humans by bites of Aedes aegypti and Aedes albopictus mosquitoes. Although, studies have been undertaken to combat the disease still there are no effective strategies like vaccines or antivirals against it. Therefore it is essential to understand the virus and host interaction to overcome this hurdle. In this study two host factors MK2 and MK3 have been taken into consideration to see how they regulate the multiplication of the virus. The in vitro experiments demonstrated that inhibition of MK2 and MK3 restricted viral infection Further, it was observed that this is due to the blocking of lamellipodium formation by modifying the factors involved in the actin cytoskeleton remodeling pathway that is responsible for releasing the virus from the infected cells. Besides, decreased disease score as well as better survivability was noticed in the in vivo experiments with mice. Therefore, MK2 and MK3 could be considered as the key targets for controlling CHIKV infection. family Togaviridae and transmitted to humans by Aedes mosquitoes(1). Three CHIKV 92 genotypes, namely West African, East Central South African and Asian have been identified. 93 The incubation period ranges from two to five days following which symptoms such as fever (up 94 to 40°C), petechial or maculopapular rash of the trunk and arthralgia affecting multiple joints 95 develop(2-4). CHIKV is a small (60-70nm diameter), spherical, enveloped, positive sense single-stranded RNA 97 (~12Kb) virus (5-7). Its genomic organization is 5′-cap-nsP1-nsP2-nsP3-nsP4-(junction region)- Raw data sets were extracted from all text files after scanning the TIFF files. These raw data sets 160 were analyzed separately using the GeneSpring GX12.0 software (Agilent Technologies, USA) 161 followed by differential gene expression and cluster analysis. Differential gene expression 162 analyses were performed by using standard fold change cut off >=2.0 and >=10.0 against IS The CHIKV-infected cell culture supernatants were collected at 18 hpi and subjected to plaque 198 assay according to the procedure mentioned earlier (29). Immunofluorescence staining 200 Immunofluorescence staining was carried out using the procedures described earlier ( Vero cells were infected with CHIKV as described above and CMPD1 (50µM) was added at 1hr 235 interval upto 11hrs to the infected cells in different dishes. Thereafter, cell culture supernatants 236 of all the samples were harvested at 15hpi and plaque assay was carried out for estimating viral 237 titer. The mice related experiments were performed as per CPCSEA guidelines and were approved by 240 the IAEC committee. Around 10-14 days old male C57BL/6 mice (n=5) were injected 241 subcutaneously with 1x10 6 particles of IS in DMEM. At 3hpi, mice were fed with CMPD1 at a 242 concentration of 5mg/kg of body weight and continually fed at every 24hr-interval up to3 days. All mice were sacrificed on the fourth day; blood samples were harvested from mock, infected with Dunnet post-hoc test was used to compare the differences between the groups. In all the 266 tests, p value < 0.05 was considered to be statistically significant. in Fig 1B. A pie-chart was constructed using the Panther software to annotate these genes into 286 different biological processes, and it was observed that majority of the modulated genes 287 belonged to the pathways involved in different cellular processes ( Fig 1C) . Moreover, 720 genes 288 were differently regulated by IS alone as depicted by the Venn diagram constructed through the 289 Gene Venn software in (Fig 1D and 1E ). Out of these 720 genes, few selected genes were 290 functionally annotated into different host cellular pathways as shown in "S1 together as compared to control as shown in Fig 2A and 2B (left and right panels respectively). As the expression of nsP2 was increased after siRNA treatment, plaque assay was performed to 308 assess the effect of MK2 and/or MK3 down-regulation in viral progeny formation. Interestingly, 309 it was observed that the viral titers were reduced by 58% for PS strain and 53% for IS strain as 310 shown in Fig 2C and 2D . Therefore, it can be suggested that MK2 and MK3 altogether affects 311 CHIKV progeny release without affecting viral protein synthesis. The MAPK-activated protein kinases MAPKAPK3 (MK3) and MAPKAPK2 (MK2) In order to confirm the involvement of actin fibers in CHIKV progeny release, Vero cells were 373 virus infected and drug treated as mentioned above and cells were fixed at 18 hpi. Thereafter, 374 phalloidin staining was carried out to stain actin fibers in cells as it has been reported that 375 fluorescent dye-labeled phalloidin stains only the actin fibers, but not the monomers (38). Phalloidin staining was found to be more prominent in infected cells without CMPD1 treatment 377 and was more diffusely stained in CMPD1 treated infected cells. Furthermore, the expression 378 pattern of CHIKV E2 protein was unchanged in both the samples as shown in Fig 4D. Taken 379 together, the results depict that CHIKV utilizes the actin polymerization process for its progeny 380 release through activation of MK2/MK3; however CMPD1 abrogates the whole process by 381 inhibiting MK2/3 activation. In order to assess the bio-availability of a drug/inhibitor, computer models have been used as a 384 valid alternative to experimental procedures for prediction of ADME (Absorption, Distribution, Metabolism and Excretion) parameters (39). The SwissADME Web tool (www.swissadme.ch) is 386 one such tool which enables the computation of key physicochemical, pharmacokinetic, drug-387 like and related parameters for one or multiple molecules (40). Hence, the bioavailability of 388 CMPD1 was predicted through the SwissADME tool and it was found that CMPD1 has high GI 389 (Gastro Intestinal) absorption with a bioavailability score of 0.55 as shown in "S2 Table" . Table" . Moreover, from the survival curve analysis as shown in Figure 409 5e, there was 100% mortality of the untreated CHIKV infected mice after 8 days post infection. In contrast, no mortality was observed for the CMPD1 treated CHIKV infected mice even after 8 411 days post infection. The data suggests that CMPD1 shows anti-CHIKV activity in vivo. In order to assess the efficacy of CMPD1 against other viruses like HSV-1 and SARS-CoV-2, CHIKV is now considered as a major public health concern. 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