Jyoti Rana

Mapping interactions of Chikungunya virus nonstructural proteins.

The four nonstructural proteins (nsPs1-4) of Chikungunya virus (CHIKV) play important roles involving enzymatic activities and specific interactions with both viral and host components, during different stages of viral pathogenesis. Elucidation of the presence and/or absence of interactions among nsPs in a systematic manner is thus of scientific interest. In the current study, each pair-wise combination among the four nonstructural proteins of CHIKV was systematically analyzed for possible interactions. Six novel protein interactions were identified for CHIKV, using systems such as yeast two-hybrid, GST pull down and ELISA, three of which have not been previously reported for the genus Alphavirus. These interactions form a network of organized associations that suggest the spatial arrangement of nonstructural proteins in the late replicase complex. The study identified novel interactions as well as concurred with previously described associations in related alphaviruses.

Intraviral Protein interactions of Chandipura Virus

Chandipura virus (CHPV) is an emerging rhabdovirus responsible for several outbreaks of fatal encephalitis among children in India. The characteristic structure of the virus is a result of extensive and specific interplay among its five encoded proteins. The revelation of interactions among CHPV proteins can help in gaining insight into viral architecture and pathogenesis. In the current study, we carried out comprehensive yeast two-hybrid (Y2H) analysis to elucidate intraviral protein-protein interactions. All of the interactions identified by Y2H were assessed for reliability by GST pull-down and ELISA. A total of eight interactions were identified among four viral proteins. Five of these interactions are being reported for the first time for CHPV. Among these, the glycoprotein (G)-nucleocapsid (N) interaction could be considered novel, as this has not been reported for any members of the family Rhabdoviridae. This study provides a framework within which the roles of the identified protein interactions can be explored further for understanding the biology of this virus at the molecular level.

Network mapping among the functional domains of Chikungunya virus nonstructural proteins

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. Proteins 2014; 82:2403–2411.

Host–pathogen interactome analysis of Chikungunya virus envelope proteins E1 and E2

Abstract The envelope proteins of Chikungunya virus (CHIKV) are known to play crucial roles in viral infection and spread. Although the role of envelope proteins in viral infection has been studied, the cellular interactors of these proteins are still elusive. In the present study, the ectodomains of CHIKV envelope proteins (E1 and E2) have been used for a high throughput yeast two-hybrid (Y2H) screening to identify the interacting host protein partners. Following a comparative analysis between the viral–host protein interaction data generated from Y2H and computational approach, five host proteins interacting with E1 and three host proteins interacting with E2 common to both datasets were identified. These associations were further verified independently by pull down and protein interaction ELISA. The identified interactions shed light on the possible cellular machinery that CHIKV might be employing during viral entry, trafficking, and evasion of immune system.

Predicting the host protein interactors of Chandipura virus using a structural similarity–based approach

Chandipura virus (CHPV), alike other pathogens, exploits the cellular infrastructure of their hosts through complex network of interactions for successful infection. CHPV being a recently emerged pediatric encephalitic virus, the mechanisms involved in the establishment of viral persistence are still ill defined. Because the protein interface between CHPV and its host provides one means by which the virus invades and seize control of their human host machinery, the authors in this study have employed computational methods to create a network of putative protein-protein interactions between CHPV and its human host to shed light on the hitherto less-known CHPV biology. On the basis of the 2105 potential interactions predicted among 1650 human proteins and the five proteins of CHPV, the authors decipher the probable mode by which the virus manipulates the biological pathways of its host toward its own end and replicates while evading the immune system. Identification of such conserved set of putative interactions that allow the virus to take control of the host has the potential to deepen our understanding of the virus-specific remodeling processes of the host cell and illuminate new arenas of disease intervention.

Elucidating the Interacting Domains of Chandipura Virus Nucleocapsid Protein

The nucleocapsid (N) protein of Chandipura virus (CHPV) plays a crucial role in viral life cycle, besides being an important structural component of the virion through proper organization of its interactions with other viral proteins. In a recent study, the authors had mapped the associations among CHPV proteins and shown that N protein interacts with four of the viral proteins: N, phosphoprotein (P), matrix protein (M), and glycoprotein (G). The present study aimed to distinguish the regions of CHPV N protein responsible for its interactions with other viral proteins. In this direction, we have generated the structure of CHPV N protein by homology modeling using SWISS-MODEL workspace and Accelrys Discovery Studio client 2.55 and mapped the domains of N protein using PiSQRD. The interactions of N protein fragments with other proteins were determined by ZDOCK rigid-body docking method and validated by yeast two-hybrid and ELISA. The study revealed a unique binding site, comprising of amino acids 1–30 at the N terminus of the nucleocapsid protein (N1) that is instrumental in its interactions with N, P, M, and G proteins. It was also observed that N2 associates with N and G proteins while N3 interacts with N, P, and M proteins.

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

Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is considered to be the master regulator of viral RNA replication and host responses generated during viral infection. This protein has two main functional domains: an N-terminal domain which exhibits NTPase, RNA triphosphatase and helicase activities and a C-terminal protease domain. Understanding how CHIKV nsP2 interacts with its host proteins is essential for elucidating all the required processes for viral replication and pathogenesis along with the identification of potential targets for antiviral therapy. In current study yeast two-hybrid (Y2H) screening of a human fetal brain cDNA library was performed using nsP2 protein as bait. The analysis identified seven host proteins (CCDC130, CPNE6, POLR2C, MAPK9, EIF4A2, EEF1A1 and EIF3I) as putative interactors of CHIKV nsP2 which were selected for further analysis based on their roles in host cellular machinery. The gene ontology analysis indicates that these proteins are mainly involved in apoptosis, transcription and translational mechanism of host cell. Domain mapping of nsP2 revealed that these associations are not random connections but instead they have functional significance. Further studies to identify the amino acid residues and their chemical interactions that may help in opening new possibilities for preventing these interactions, thus reducing chances of chikungunya infection were performed. This study expands the understanding of CHIKV-host interactions and is important for rational approaches of discovering new antiviral agents.

Neuroinvasion by Chandipura virus.

Chandipura virus (CHPV) is an arthropod borne rhabdovirus associated with acute encephalitis in children below the age of 15 years in the tropical states of India. Although the entry of the virus into the nervous system is among the crucial events in the pathogenesis of CHPV, the exact mechanism allowing CHPV to invade the central nervous system (CNS) is currently poorly understood. In the present review, based on the knowledge of host interactors previously predicted for CHPV, along with the support from experimental data available for other encephalitic viruses, the authors have speculated the various plausible modes by which CHPV could surpass the blood-brain barrier and invade the CNS to cause encephalitis whilst evading the host immune surveillance. Collectively, this review provides a conservative set of potential interactions that can be employed for future experimental validation with a view to better understand the neuropathogenesis of CHPV.

Characterization of Yuhushiella sp. TD-032 from the Thar Desert and its antimicrobial activity.

During a screening program for antimicrobial compounds from underexplored habitats, a Gram-positive bacterium TD-032, was isolated from arid soil, Thar Desert (India), and analyzed for its morphological, physicochemical, and antimicrobial properties. The 16S ribosomal DNA (rDNA) sequence of the isolate was further studied for the novelty of γ-hyper variable region. TD-032 was grown in large-scale culture, and aqueous and organic solvent extracts analyzed for antimicrobial activity. Culture characteristics showed a lack of diffusible and melanoid pigments. The morphological features were pale yellow aerial mycelium colony color with brownish yellow substrate mycelium and leathery texture. The isolate could grow at 1% concentration of sodium chloride, temperature of 40΀C, and a wide range of pH (7.0-12.0). An evaluation for extracellular enzymatic activities showed secretion of gelatinase(s), cellulase(s), and lipase(s). The γ-hyper variable region of 16S rDNA sequence of TD-032 showed 98.33% relatedness to Yuhushiella deserti, indicating a potential new species. Aqueous and ethyl acetate extracts showed antimicrobial activity against Gram-positive and Gram-negative bacteria inclusive clinical isolates. Inhibition of both test bacteria suggests that TD-032 produces a broad spectrum of antimicrobial substances.