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Mosquito-borne viral diseases like chikungunya and dengue infections can cause severe illness and have become major public health concerns. Chikungunya virus (CHIKV) and dengue virus (DENV) infections share similar primary clinical manifestations and are transmitted by the same vector. Thus, the probability of their coinfection gets increased with more severe clinical complications in the patients. The present study was undertaken to elucidate the common human interacting partners of CHIKV and DENV proteins during coinfection. The viral–host protein–protein interactome was constructed using Cytoscape. Subsequently, significant host interactors were identified during coinfection. The network analysis elucidated 57 human proteins interacting with both CHIKV and DENV, represented as hub-bottlenecks. The functional and biological analyses of the 40 hub-bottlenecks revealed that they are associated with phosphoinositide 3-kinases (PI3K)/AKT, p53 signaling pathways, regulation of cell cycle and apoptosis during coinfection. Moreover, the molecular docking analysis uncovered the tight and robust binding of selected hub-bottlenecks with CHIKV/DENV proteins. Additionally, 23 hub-bottlenecks were predicted as druggable candidates that could be targeted to eradicate the host–viral interactions. The elucidated common host binding partners during DENV and CHIKV coinfection as well as indicated approved drugs can support the therapeutics development. [ABSTRACT FROM AUTHOR]

• AUD is a multifunctional domain of nsP3 essential for Chikungunya virus life cycle. • The human interactors of CHIKV AUD were identified utilizing yeast two hybrid system. • SNAPIN and N4BP2L2 interaction with CHIKV AUD was validated by pull down assay. • Key residues involved in SNAPIN and N4BP2L2 association with AUD were predicted. The life-threatening re-emerged chikungunya virus (CHIKV) can cause an epidemic outbreak and still has no vaccine available so far. Alphavirus unique domain (AUD) of CHIKV nsP3 is a multifunctional domain that remains conserved among alphaviruses and is critical for CHIKV replication. The understanding of AUD-host protein-protein interactions and their association with the cellular processes concerning CHIKV infection are not well studied. In the current study, the protein-protein interactions of AUD and its human host were elucidated by screening of universal human cDNA library using yeast two-hybrid system. The chosen interactions were further validated by GST pull-down assay, and their network mapping was analyzed. The study revealed that the identified interactors are linked with the vesicle trafficking and transcription corepressor activities. Further, the interfacial residues of interactions between viral and host proteins were predicted, which will further provide the new platform to develop novel antivirals. [ABSTRACT FROM AUTHOR]

One of the most important rapidly emerging mosquito-borne alphavirus is Chikungunya virus (CHIKV). There is a necessity to develop anti-CHIKV therapeutics, as neither antiviral drug nor vaccines have been licensed yet. Several CHIKV proteins are being studied worldwide, but non-structural protein 3 (nsP3) has been less explored. This protein consists of three domains: macrodomain, alphavirus unique domain (AUD) and hypervariable region (HVR). The proline-rich regions of HVR contain SRC homology 3 (SH3)-binding domain which is essential for its functionality. Interaction of these motifs with host amphiphysin protein is crucial for viral RNA replication. Restricting the interactions of HVR could lead to inhibition of viral life cycle. Therefore, the present study focuses on purification of HVR protein and its structural and functional assay for therapeutic intervention in future use. In order to obtain purified protein, HVR region was amplified from TOPO clones of nsP3 of IND-06-Guj strain and cloned into expression vector. Expression and solubilization of the protein were optimized at various conditions of salt, detergent and imidazole before purification. The soluble recombinant HVR (His-HVR) protein was purified using affinity chromatography. Purified protein was analyzed for structural studies and functional assays. Circular dichroism of His-HVR protein was performed for structural study, and it was observed that it consists of mostly random coils. For functional assay, co-pull down of His-HVR protein was performed with endogenous amphiphysin-I protein of N2a cells and was analyzed using Western blotting. This purified protein obtained could be used as a potential target reagent for novel therapeutic interventions in the future. [ABSTRACT FROM AUTHOR]