Soma Chattopadhyay

Heat Shock Protein 90 Positively Regulates Chikungunya Virus Replication by Stabilizing Viral Non-Structural Protein nsP2 during Infection

Background The high morbidity and socio-economic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV) emphasize the need to understand the biology of the virus for developing effective antiviral therapies. Methods and Findings In this study, an attempt was made to understand the molecular mechanism involved in Heat shock protein 90 (Hsp90) mediated regulation of CHIKV infection in mammalian cells using CHIKV prototype strain (S 27) and Indian outbreak strain of 2006 (DRDE-06). Our results showed that Hsp90 is required at a very early stage of viral replication and Hsp90 inhibitor Geldanamycin (GA) can abrogate new virus particle formation more effectively in the case of S 27 than that of DRDE-06. Further analysis revealed that CHIKV nsP2 protein level is specifically reduced by GA treatment as well as HSP90-siRNA transfection; however, viral RNA remains unaltered. Immunoprecipitation analysis showed that nsP2 interacts with Hsp90 during infection; however this interaction is reduced in the presence of GA. In addition, our analysis on Hsp90 associated PI3K/Akt/mTOR signaling pathway demonstrated that CHIKV infection stabilizes Raf1 and activates Hsp90 client protein Akt, which in turn phosphorylates mTOR. Subsequently, this phosphorylation leads to the activation of two important downstream effectors, S6K and 4EBP1, which may facilitate translation of viral as well as cellular mRNAs. Hence, the data suggests that CHIKV infection is regulated by Hsp90 associated Akt phosphorylation and DRDE-06 is more efficient than S 27 in enhancing the activation of host signaling molecules for its efficient replication and virus production. Conclusion Hsp90 positively regulates Chikungunya virus replication by stabilizing CHIKV-nsP2 through its interaction during infection. The study highlights the possible molecular mechanism of GA mediated inhibition of CHIKV replication and differential effect of this drug on S 27 and DRDE-06, which will be informative for developing effective anti-CHIKV therapies in future.

High rates of co-infection of Dengue and Chikungunya virus in Odisha and Maharashtra, India during 2013.

Dengue viral (DENV) infection is endemic in different parts of India and because of similar primary signs and symptoms, Chikungunya virus (CHIKV) is mostly undiagnosed. Hence, we investigated 204 suspected Dengue cases in a hospital based cross-sectional study in Odisha, India in 2013. It was observed that 50 samples were positive for DENV only, 28 were positive for CHIKV only and interestingly, 28 patients were co-infected with both DENV and CHIKV. Additionally, a total of 18 confirmed Dengue samples from Maharashtra, India were screened for CHIKV and out of those, 15 were co-infected. All CHIKV strains were of East Central South African (ECSA) type and serotype 2 (genotype IV) was predominant in the DENV samples. Additionally, Dengue serotype 1 and 3 were also detected during this time. Further, sequence analysis of E1 gene of CHIKV strains revealed that two substitution mutations (M269V and D284E) were observed in almost 50% strains and they were from co-infected patients. Similarly, sequence analysis of C-prM gene showed the presence of five substitution mutations, (G70S, L72F, N90S, S93N and I150L) in all serotype 1 and two consistent mutations (A101V and V112A) in serotype 2 Dengue samples. Together, it appears that a significantly high number of dengue patients (43, 44.8%) were co-infected with DENV and CHIKV during this study. This emphasizes the need of a routine diagnosis of CHIKV along with DENV for febrile patients. This will be useful in early and proper recognition of infecting pathogen to study the correlation of clinical symptoms with single or co-infection which will ultimately help to implement proper patient care in future.

The two helicases of herpes simplex virus type 1 (HSV-1).

Herpes simplex virus type 1 (HSV-1) encodes two helicases both of which are essential for viral DNA synthesis. UL9 binds specifically to the origins of replication and is believed to initiate DNA replication at one of three origins of replication located in the HSV-1 genome. The heterotrimeric helicase-primase complex, encoded by the UL5, UL8 and UL52 genes, is believed to unwind duplex viral DNA at replication forks and to prime lagging strand synthesis. Functional analyses of UL9 and the helicase-primase complex will be discussed with attention to the roles these proteins play during HSV-1 replication.

A Novel 2006 Indian Outbreak Strain of Chikungunya Virus Exhibits Different Pattern of Infection as Compared to Prototype Strain

Background The recent re-emergence of Chikungunya virus (CHIKV) in India after 32 years and its worldwide epidemics with unprecedented magnitude raised a great public health concern. Methods and Findings In this study, a biological comparison was carried out between a novel 2006 Indian CHIKV outbreak strain, DRDE-06 and the prototype strain S-27 in mammalian cells in order to understand their differential infection pattern. Results showed that S-27 produced maximum number of progenies (2.43E+06 PFU/ml) at 20 to 24 hours post infection whereas DRDE-06 produced more than double number of progenies around 8 hours post infection in mammalian cells. Moreover, the observation of cytopathic effect, detection of viral proteins and viral proliferation assay confirmed the remarkably faster and significantly higher replication efficiency of DRDE-06. Moreover, our mutational analysis of whole genome of DRDE-06 revealed the presence of nineteen mutations as compared to S-27, whereas the analysis of 273 global isolates showed the consistent presence of fifteen out of nineteen mutations in almost all outbreak isolates. Further analysis revealed that ∼46% of recent outbreak strains including DRDE-06 do not contain the E1-A226V mutation which was earlier shown to be associated with the adaptation of CHIKV in a new vector species, Aedes albopictus. Conclusions A novel 2006 Indian CHIKV outbreak strain, DRDE-06 exhibits different pattern of infection as compared to prototype strain, S-27. This might be associated to some specific mutations observed in genome wide mutational analysis in DRDE-06 which emphasizes the need of future experimental investigation.

Inhibition of Chikungunya Virus Replication by 1-[(2-Methylbenzimidazol-1-yl) Methyl]-2-Oxo-Indolin-3-ylidene] Amino] Thiourea(MBZM-N-IBT)

Chikungunya virus (CHIKV) infection is one of the most challenging human Arboviral infections with global significance and without any specific antiviral. In this investigation, 1-[(2-methylbenzimidazol-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT) was synthesised as a molecular hybrid of 2-methyl benzimidazole and isatin-β-thiosemicarbazone and its anti-CHIKV property was evaluated. The release of infectious virus particles was calculated by plaque assay, expression profile of viral RNA was estimated by RT-PCR and viral protein profiles were assessed by Western blot and FACS analyses. The safety index of MBZM-N-IBT was found to be >21. The CHIKV infectious viral particle formation was abrogated around 76.02% by MBZM-N-IBT during infection in mammalian system and the viral RNA synthesis was reduced by 65.53% and 23.71% for nsP2 and E1 respectively. Surprisingly, the viral protein levels were reduced by 97% for both nsP2 and E2. In the time-of-addition experiment it abrogated viral infection at early as well as late phase of viral life cycle, which indicates about multiple mechanisms for its anti-CHIKV action. In silico analysis justified development of MBZM-N-IBT with good affinities for potential target proteins of CHIKV and related virus. With predictions of good drug-likeness property, it shows potential of a drug candidate which needs further experimental validation.

Direct Interaction between the N- and C-Terminal Portions of the Herpes Simplex Virus Type 1 Origin Binding Protein UL9 Implies the Formation of a Head-to-Tail Dimer

ABSTRACT UL9, a superfamily II helicase, is a multifunctional protein required for herpes simplex virus type 1 replication in vivo. Although the C-terminal 317-amino-acid DNA binding domain of UL9 exists as a monomer, the full-length protein behaves as a dimer in solution. Thus, it has been assumed that the N-terminal 534 residues contain a region necessary for efficient dimerization and that UL9 dimers are in a head-to-head configuration. We recently showed, however, that residues in the N terminus could modulate the inhibitory properties of UL9 by decreasing the DNA binding ability of the C terminus (S. Chattopadhyay and S. K. Weller, J. Virol. 80:4491-4500, 2006). We suggested that a direct interaction between the N- and C-terminal portions of UL9 might exist and serve to modulate the DNA binding activities of the C terminus. In this study, we used a coimmunoprecipitation assay to show that the N-terminal portion of UL9 can indeed directly interact with the C terminus. A series of truncation mutant proteins were used to show that a region in the N terminus between residues 293 and 321 is necessary for efficient interaction. Similarly, a region in the C terminus between residues 600 and 800 is required for this interaction. The simplest model to explain these data is that UL9 dimers are oriented in a head-to-tail arrangement in which the N terminus is in contact with the C terminus.

In silico analysis of MHC-I restricted epitopes of Chikungunya virus proteins: Implication in understanding anti-CHIKV CD8+ T cell response and advancement of epitope based immunotherapy for CHIKV infection

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus, responsible for acute febrile infection. The high morbidity and socio-economic loss associated with the recent CHIKV epidemics worldwide have raised a great public health concern and emphasize the need to study the immunological basis of CHIKV infection to control the disease. MHC-I restricted CD8(+) T cell response represent one of the major anti-viral immune responses. Accordingly, it is essential to have a detailed understanding towards CHIKV specific MHC-I restricted immunogenic epitopes for anti-viral CD8(+) CTL immunogenicity. In the present study, a computational approach was used to predict the conserved MHC-I epitopes for mouse haplotypes (H2-Db and H2-Dd) and some alleles of the major HLA-I supertypes (HLA-A2, -A3, -A24, -B7, -B15) of all CHIKV proteins. Further, an in-depth computational analysis was carried out to validate the selected epitopes for their nature of conservation in different global CHIKV isolates to assess their binding affinities to the appropriate site of respective MHC-I molecules and to predict anti-CHIKV CD8(+) CTL immunogenicity. Our analyses resulted in fifteen highly conserved epitopes for H2-Db and H2-Dd and fifty epitopes for different HLA-I supertypes. Out of these, the MHC-I epitopes VLLPNVHTL and MTPERVTRL were found to have highest predictable CTL immunogenicities and least binding energies for H2-Db and H2-Dd, whereas, for HLA-I, the epitope FLTLFVNTL was with the highest population coverage, CTL immunogenicity and least binding energy. Hence, our study has identified MHC-I restricted epitopes that may help in the advancement of MHC-I restricted epitope based anti-CHIKV immune responses against this infection and this will be useful towards the development of epitope based anti-CHIKV immunotherapy in the future. However, further experimental investigations for cross validation and evaluation are warranted to establish the ability of epitopes to induce CD8(+) T cell mediated immune responses.

Mammalian non-classical major histocompatibility complex I and its receptors: Important contexts of gene, evolution, and immunity

The evolutionary conserved, less-polymorphic, nonclassical major histocompatibility complex (MHC) class I molecules: Qa-1 and its human homologue human leukocyte antigen-E (HLA-E) along with HLA-F, G and H cross-talk with the T-cell receptors and also interact with natural killer T-cells and other lymphocytes. Moreover, these nonclassical MHC molecules are known to interact with CD94/NKG2 heterodimeric receptors to induce immune responses and immune regulations. This dual role of Qa-1/HLA-E in terms of innate and adaptive immunity makes them more interesting. This review highlights the new updates of the mammalian nonclassical MHC-I molecules in terms of their gene organization, evolutionary perspective and their role in immunity.

Development and characterization of monoclonal antibody against non-structural protein-2 of Chikungunya virus and its application.

The recent epidemics of Chikungunya viruses (CHIKV) with unprecedented magnitude and unusual clinical severity have raised a great public health concern worldwide, especially due to unavailability of vaccine or specific therapy. This emphasizes the need to understand the biological processes of this virus in details. Although CHIKV associated research has been initiated, the availability of CHIKV specific reagents for in-depth investigation of viral infection and replication are scanty. For Alphavirus replication, non-structural protein 2 (nsP2) is known to play a key regulatory role among all other non-structural proteins. The current study describes the development and characterization of nsP2 specific monoclonal antibody (mAb) against a synthetic peptide of CHIKV. Reactivity and efficacy of this mAb have been demonstrated by ELISA, Western blot, Flow cytometry and Immunofluorescence assay. Time kinetic study confirms that this mAb is highly sensitive to CHIKV-nsP2 as this protein has been detected very early during viral replication in infected cells. Homology analysis of the selected epitope sequence reveals that it is conserved among all the CHIKV strains of different genotypes, while analysis with other Alphavirus sequences shows that none of them are 100% identical to the epitope sequence. Moreover, using the mAb, three isoforms of CHIKV-nsP2 have been detected in 2D blot analysis during infection in mammalian cells. Accordingly, it can be suggested that the mAb reported in this study can be a sensitive and specific tool for experimental investigations of CHIKV replication and infection.

Regulation of Viral Replication, Apoptosis and Pro-Inflammatory Responses by 17-AAG during Chikungunya Virus Infection in Macrophages.

Chikungunya virus (CHIKV) infection has re-emerged as a major public health concern due to its recent worldwide epidemics and lack of control measures. Although CHIKV is known to infect macrophages, regulation of CHIKV replication, apoptosis and immune responses towards macrophages are not well understood. Accordingly, the Raw264.7 cells, a mouse macrophage cell line, were infected with CHIKV and viral replication as well as new viral progeny release was assessed by flow cytometry and plaque assay, respectively. Moreover, host immune modulation and apoptosis were studied through flow cytometry, Western blot and ELISA. Our current findings suggest that expression of CHIKV proteins were maximum at 8 hpi and the release of new viral progenies were remarkably increased around 12 hpi. The induction of Annexin V binding, cleaved caspase-3, cleaved caspase-9 and cleaved caspase-8 in CHIKV infected macrophages suggests activation of apoptosis through both intrinsic and extrinsic pathways. The pro-inflammatory mediators (TNF and IL-6) MHC-I/II and B7.2 (CD86) were also up-regulated during infection over time. Further, 17-AAG, a potential HSP90 inhibitor, was found to regulate CHIKV infection, apoptosis and pro-inflammatory cytokine/chemokine productions of host macrophages significantly. Hence, the present findings might bring new insight into the therapeutic implication in CHIKV disease biology.