Intraviral protein interactions of Chandipura virus

Kumar, Kapila; Rana, Jyoti; Rajasekharan, Sreejith; Gabrani, Reema; Sharma, Sanjeev; Gupta, Amita; Chaudhary, Vijay; Gupta, Sanjay

doi:10.1007/s00705-012-1389-5

Cited by 13 publications

(17 citation statements)

References 41 publications

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“…ing stringent Y2H system. This system is a useful tool to study direct protein-protein interactions and has been used widely for intraviral and viral-host interactome analysis e.g., vaccinia virus (Zhang et al, 2009), Epstein-Barr virus (Calderwood et al, 2007), poxvirus (Vliet et al, 2009, dengue virus (Mairiang et al, 2013), hepatitis E virus (Geng et al, 2013), hepatitis C virus (Dolan et al, 2013), Chandipura virus (Rajasekharan et al, 2015;Kumar et al, 2012) and chikungunya virus (Sreejith et al, 2012;Rana et al, 2014;Dudha et al, 2014). The viral protein nsP2 is an important candidate to study CHIKV-host interactions because it represents a potential target for designing novel antiviral therapy as it is a viral protease required for maturation of nsPs for conversion of early replicase complex into late replicase complex and also for inhibition of host responses triggered during viral infection.…”

Section: Discussionmentioning

confidence: 99%

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

Rana¹,

Gulati²,

Rajasekharan³

et al. 2017

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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.

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Section: Discussionmentioning

confidence: 99%

“…Following induction, the induced cells were harvested and lysed using lysis buffer (IBA-GmbH, Germany). The lysates (soluble protein fraction) were used to perform pull down and protein interaction ELISA using protocols described in our earlier works (Sreejith et al, 2012;Kumar et al, 2011Kumar et al, , 2012.…”

Section: Methodsmentioning

confidence: 99%

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

Rana¹,

Gulati²,

Rajasekharan³

et al. 2017

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“…Many of these new vectors have been used extensively for cloning Mtb toxin and anti-toxin genes and to carry out functional studies with the co-transformants by differential expression using arabinose and tetracycline [20]. The vectors mentioned above have also been used to clone and express several Chandipura virus proteins [21].…”

Section: Discussionmentioning

confidence: 99%

Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

et al. 2014

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Herein, we describe a novel cloning strategy for PCR-amplified DNA which employs the type IIs restriction endonuclease BsaI to create a linearized vector with four base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in presence of a single dNTP to create vector-compatible four base-long overhangs. Notably, the insert preparation does not require any restriction enzyme treatment. The BsaI sites in the vector are oriented in such a manner that upon digestion with BsaI, a stuffer sequence along with both BsaI recognition sequences is removed. The sequence of the four base-long overhangs produced by BsaI cleavage were designed to be non-palindromic, non-compatible to each other. Therefore, only ligation of an insert carrying compatible ends allows directional cloning of the insert to the vector to generate a recombinant without recreating the BsaI sites. We also developed rapid protocols for insert preparation and cloning, by which the entire process from PCR to transformation can be completed in 6–8 h and DNA fragments ranging in size from 200 to 2200 bp can be cloned with equal efficiencies. One protocol uses a single tube for insert preparation if amplification is performed using polymerases with low 3′-exonuclease activity. The other protocol is compatible with any thermostable polymerase, including those with high 3′-exonuclease activity, and does not significantly increase the time required for cloning. The suitability of this method for high-throughput cloning was demonstrated by cloning batches of 24 PCR products with nearly 100% efficiency. The cloning strategy is also suitable for high efficiency cloning and was used to construct large libraries comprising more than 108 clones/µg vector. Additionally, based on this strategy, a variety of vectors were constructed for the expression of proteins in E. coli, enabling large number of different clones to be rapidly generated.

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“…The predicted interactions are highlighted on the basis of their functional relevance during CHPV infection and prioritized based on the interaction data of other RNA viruses like vesicular stomatitis virus (VSV), Japanese encephalitis virus , West Nile virus , and Influenza A virus . Together with the knowledge of intraviral protein interactions of CHPV previously reported by the authors (Kumar et al ., ), the current study puts forth a platform for the better understanding of CHPV biology.…”

Section: Introductionmentioning

confidence: 91%

“…In this approach, the human proteins with defined structure and known interactions are mapped to CHPV proteins for structural similarity. The authors have previously adopted this approach to decipher the viral-host protein interface in chikungunya virus-mediated sickness (Rana et al, 2012). This approach has also been used earlier for predicting the interactions among HIV-human and dengue viru0073-human proteins (Doolittle & Gomez, 2010.…”

Section: Introductionmentioning

confidence: 99%

Predicting the host protein interactors ofChandipura virususing a structural similarity-based approach

et al. 2013

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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.

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Intraviral protein interactions of Chandipura virus

Cited by 13 publications

References 41 publications

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

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

Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

Predicting the host protein interactors ofChandipura virususing a structural similarity-based approach

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