Deciphering the human cellular interactors of alphavirus unique domain of chikungunya virus

Highlights

• 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.

Abstract

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.

Introduction

Chikungunya virus (CHIKV), a re-emerging arbovirus, belongs to Togaviridae family and Alphavirus genus. It causes arthralgia, myalgia, rash, fever, and headache that lasts up to two weeks during acute infection, however, prolonged joint pains often last from months to years (Galán-Huerta et al., 2015; Stelitano et al., 2019). Neurological disorders, ocular and cardiovascular complications reported to be associated with CHIKV infection have turned out to be more life-threatening (Galán-Huerta et al., 2015; Silva and Dermody, 2017). The outbreaks of CHIKV have been reported worldwide in tropical as well as temperate regions across Africa, Asia, Europe, and America. The severity of this disease due to the increasing global climate changes, convenient for its vector propagation and transmission to vertebrates, has become a serious concern (Ryan et al., 2019). It emphasises the urgency to develop an effective therapeutic treatment.

CHIKV has positive-sense, single-stranded RNA genome with two open reading frames (ORFs) encoding structural and non-structural proteins (nsPs) at 3′ and 5′ ends, respectively. All the nsPs (nsP1 to nsP4) are mainly required for replication and transcription i.e. synthesis of genomic and sub-genomic RNAs (Wong and Chu, 2018; Stelitano et al., 2019). All the nsPs essential for viral replication have different biological attributes; nsP1 is a viral capping enzyme; nsP2 has protease activity; nsP4 is a RNA dependent RNA polymerase, but the function of nsP3 is still elusive (Wong and Chu, 2018; Rabelo et al., 2020). Mutational studies of nsP3 have revealed its presence is critical for CHIKV replication (Meshram et al., 2018; Mutso et al., 2018; Gao et al., 2019). The nsP3 has three domains: N-terminal macro-domain, alphavirus unique domain (AUD) and the C-terminal hypervariable region (HVR) (Rabelo et al., 2020). AUD is a multifunctional, zinc-binding domain which remains conserved across alphaviruses (Götte et al., 2018). AUD plays a critical role in the viral life cycle specifically in genome replication and the synthesis of sub-genomic RNAs. Studies have reported that the mutant AUD effectively perturbs the translation and sub-genomic RNA synthesis leading to the reduction in levels of structural proteins and viral assembly. Moreover, the AUD mutants successfully hampered the sub-cellular localization of CHIKV nsP3 that led to the defective viral assembly and inhibited CHIKV infection. Thus, it emphasised the vital role of nsP3 in CHIKV life cycle (Gao et al., 2019).

During CHKIV infection, various cellular factors and processes like apoptosis, signalling pathways, interferons, stress granules, and autophagy get activated in response to infection, but the virus adopts evasion strategies and hijacks these cellular processes to aid its survival (Remenyi et al., 2018). The studies based on the interaction between nsP3 and the human host proteins is well documented, elucidating the association with phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) (PI3K-Akt-mTOR) pathways, Ras-GTPase-activating protein 1 (RASA1), Ras GTPase-activating protein-binding protein 2 (G3BP2), sphingosine kinase 2 (SK2) and heat shock protein 90 (Hsp90) during CHIKV replication (Fros et al., 2012; Lark et al., 2018; Wong and Chu, 2018). Moreover, the association of host G3BP proteins with HVR domain has been recently studied and observed to be critical for CHIKV replication (Meshram et al., 2018). Recently another study reported the interaction of the host four-and-a-half LIM domain protein 1 (FHL1) with the HVR domain and observed it to be essential and a key factor required for CHIKV infection and pathogenesis (Meertens et al., 2019).

Nonetheless, there is no study available in the literature that has investigated the cellular interacting partners of CHIKV AUD domain and their significance in CHIKV pathogenesis. To address this gap, the present study focuses to elucidate the cellular interactors of CHIKV AUD by screening normalized universal human cDNA library using yeast two-hybrid (Y2H) system. Few interactions were validated by GST pull-down assay to confirm the data obtained from Y2H. The proteome network and the associated cellular pathways of human proteins were analysed. Further, the interfacial amino acid residues and the type of interactions involved were analysed by docking studies.