Network mapping among the functional domains of Chikungunya virus nonstructural proteins

Abstract

Formation of virus specific replicase complex is among the most important steps that determines the fate of viral transcription and replication during Chikungunya virus (CHIKV) infection. In the present study, the authors have computationally generated a 3D structure of CHIKV late replicase complex on the basis of the interactions identified among the domains of CHIKV nonstructural proteins (nsPs) which make up the late replicase complex. The interactions among the domains of CHIKV nsPs were identified using systems such as pull down, protein interaction ELISA, and yeast two-hybrid. The structures of nsPs were generated using I-TASSER and the biological assembly of the replicase complex was determined using ZRANK and RDOCK. A total of 36 interactions among the domains and full length proteins were tested and 12 novel interactions have been identified. These interactions included the homodimerization of nsP1 and nsP4 through their respective C-ter domains; the associations of nsP2 helicase domain and C-ter domain of nsP4 with methyltransferase and membrane binding domains of nsP1; the interaction of nsP2 protease domain with C-ter domain of nsP4; and the interaction of nsP3 macro and alphavirus unique domains with the C-ter domain of nsP1. The novel interactions identified in the current study form a network of organized associations that suggest the spatial arrangement of nsPs in the late replicase complex of CHIKV.

Chikungunya Virus Overcomes Polyamine Depletion by Mutation of nsP1 and the Opal Stop Codon To Confer Enhanced Replication and Fitness

“…Because the V326M mutation was present in the as-yet-undescribed domain 3 (Fig. 5A) (18), we chose to focus on what impact this mutation would have on viral genome capping. BHK-21 cells were infected at an MOI of 0.1 with the single-mutation viruses, which have the same replication kinetics (Fig.…”

The Interplay of Viral and Host Factors in Chikungunya Virus Infection: Targets for Antiviral Strategies

“…The promiscuous alphavirus nsP2 is able to bind to not only the other nsPs but also a number of host factors. The interactions with nsPs were confirmed for CHIKV through immunoprecipitation of GST and streptavidin-tagged nsPs and their various domains, and validated via a yeast two-hybird screen and ELISA [173,174]. In addition, using computational techniques, Rana and colleagues were able to propose a spatial model of the late CHIKV replication complex [173].…”

“…The interactions with nsPs were confirmed for CHIKV through immunoprecipitation of GST and streptavidin-tagged nsPs and their various domains, and validated via a yeast two-hybird screen and ELISA [173,174]. In addition, using computational techniques, Rana and colleagues were able to propose a spatial model of the late CHIKV replication complex [173]. Bourai and colleagues performed an extensive study on the host factors interacting with the nsP2 using a yeast two-hybird screen [175].…”

Identification of potential molecular associations between chikungunya virus non-structural protein 2 and human host proteins

“…Mate & Plate-human fetal brain cDNA library (Clontech, USA) was used for Y2H screening to identify the host interactors of CHIKV nsP2 protein. The viral protein nsP2, cloned in pGBKT7 (BD) yeast expression plasmid, was transformed into Saccharomyces cerevisiae AH109 strain (generated in our earlier work; Sreejith et al, 2012) and allowed to mate with S. cerevisiae Y187 strain pre-transformed with the human fetal brain cDNA library using protocols described in our previous works (Dudha et al, 2014;Rajasekharan et al, 2015). The mated diploid clones harboring both bait (BD, coding nsP2) and prey (library plasmids/pGADT7-Rec/AD; host proteins) plasmids were initially selected on triple (TDO; SD-Trp/-Leu/-His -synthetic dropout media lacking amino acids tryptophan, leucine and histidine) followed by selection on quadruple dropout medium (QDO; SD-Trp/-Leu/-His/-Ade -synthetic dropout media lacking amino acids tryptophan, leucine and histidine, and the nitrogen base adenine) supplemented with X-α-Gal.…”

“…Further, we identified the amino acid residues that come in direct contact during these associations using protein docking studies. This study together with the knowledge of intraviral protein interaction (Sreejith et al, 2012;Rana et al, 2014) can provide molecular targets for the development of therapeutic strategies based on blockage of these associations.…”