A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein.

BACKGROUND: The influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation. METHODS: The phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging. RESULTS: The four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells. CONCLUSION: This structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.

Reprogramming of glucose metabolism in virus infected cells.

Viral infection is a kind of cellular stress that leads to the changes in cellular metabolism. Many metabolic pathways in a host cell such as glycolysis, amino acid and nucleotide synthesis are altered following virus infection. Both oncogenic and non-oncogenic viruses depend on host cell glycolysis for their survival and pathogenesis. Recent studies have shown that the rate of glycolysis plays an important role in oncolysis as well by oncolytic therapeutic viruses. During infection, viral proteins interact with various cellular glycolytic enzymes, and this interaction enhances the catalytic framework of the enzymes subsequently the glycolytic rate of the cell. Increased activity of glycolytic enzymes following their interaction with viral proteins is vital for replication and to counteract the inhibition of glycolysis caused by immune response. In this review, the importance of host cell glycolysis and the modulation of glycolysis by various viruses such as oncogenic, non-oncogenic and oncolytic viruses are presented.

Non-metabolic role of alpha-enolase in virus replication.

Viruses are extremely complex and highly evolving microorganisms; thus, it is difficult to analyse them in detail. The virion is believed to contain all the essential components required from its entry to the establishment of a successful infection in a susceptible host cell. Hence, the virion composition is the principal source for its transmissibility and immunogenicity. A virus is completely dependent on a host cell for its replication and progeny production. Occasionally, they recruit and package host proteins into mature virion. These incorporated host proteins are believed to play crucial roles in the subsequent infection, although the significance and the molecular mechanism regulated are poorly understood. One such host protein which is hijacked by several viruses is the glycolytic enzyme, Enolase (Eno-1) and is also packaged into mature virion of several viruses. This enzyme exhibits a highly flexible nature of functions, ranging from metabolic to several non-metabolic activities. All the glycolytic enzymes are known to be moonlighting proteins including enolase. The non-metabolic functions of this moonlighting protein are also highly diverse with respect to its cellular localization. Although very little is known about the virological significance of this enzyme, several of its non-metabolic functions have been observed to influence the virus replication cycle in infected cells. In this review, we have attempted to provide a comprehensive picture of the non-metabolic role of Eno-1, its significance in the virus replication cycle and to stimulate interest around its scope as a therapeutic target for treating viral pathologies.

PLCγ1­dependent invasion and migration of cells expressing NSCLC­associated EGFR mutants.

The increased tyrosine kinase activity of non­small cell lung cancer (NSCLC)­associated epidermal growth factor receptor (EGFR) mutants results in deregulated pathways that contribute to malignant cell survival, tumor progression and metastasis. Previous studies investigating lung cancer­associated EGFR have focused on the prognostic implications of receptor kinase mutations in patients with NSCLC; however, the role of EGFR mutations in tumor cell invasion and migration remains undetermined. The present study was designed to investigate the role of NSCLC­associated mutant EGFR­driven signaling pathways in cell proliferation and invasion. Non­endogenous EGFR­expressing 293 cells stably expressing EGFR mutants that are sensitive or resistant to Food and Drug Administration (FDA)­approved EGFR­targeted tyrosine kinase inhibitors (TKIs) were used in the present study. The experiments demonstrated an increased phosphorylation of phospholipase (PLC)γ1, c­Cbl, signal transducer and activator of transcription (Stat), extracellular regulated kinase (Erk)1/2, Akt, Shc and Gab1 proteins in cells expressing a mutant form, rather than the wild­type receptor. As PLCγ1 is a known regulator of metastatic development, mutant receptor­mediated PLCγ1 activation was further evaluated. To examine the effects of EGFR and PLCγ1 phosphorylation, the metastatic potential of cells expressing mutants was investigated using wound healing, Transwell cell migration and invasion assays. The inhibition of receptor phosphorylation with the 1st, 2nd and 3rd generation TKIs, gefitinib, afatinib, osimertinib, respectively, reduced PLCγ1 phosphorylation, and reduced the invasive and migratory potential of 293 cells, confirming PLCγ1 as one of the probable downstream effectors of mutant EGFR signaling. However, the PLC inhibitor, U73122, inhibited cell migration and invasion without affecting EGFR signaling and PLCγ1 phosphorylation. Notably, U73122 reduced Akt and Erk1/2 phosphorylation within 25 min of its application; however, 100% cell viability was recorded even after 48 h. Upon further investigation, proliferative signaling pathways remained active at 48 h, in accordance with cell viability. Therefore, the present study concludes that mutant receptor­mediated PLCγ1 activation may play a significant role in the migration and invasion of NSCLC tumors; however, its regulatory role in tumor cell proliferation warrants further investigation and validation in lung tumor cell lines harboring EGFR mutations.

Experimental validation of influenza A virus matrix protein (M1) interaction with host cellular alpha enolase and pyruvate kinase.

Influenza A virus, a respiratory pathogen manipulates various host cellular processes to establish a successful infection in a host. We had reported earlier the interaction of influenza A virus nucleoprotein with host glycolytic enzymes; alpha enolase and pyruvate kinase in A549 cells. Matrix protein (M1), another multifunctional protein encoded by genome segment 7 forms the inner layer of the virion and interacts with the ribonucleoprotein complex. Nucleoprotein and matrix protein, major structural components of the virion together contribute to the stability of the capsid. Thus, we have investigated the interaction of viral matrix protein with host glycolytic enzymes; alpha enolase and pyruvate kinase. Results had demonstrated differential expression of these two glycolytic enzymes in response to matrix protein and their interaction with matrix protein by in vitro binding, co-immunoprecipitation and co-localization studies. Our results confirmed that viral matrix protein interacts with host glycolytic enzymes in association with viral nucleoprotein.

Envelope proteins as antiviral drug target.

Attachment of a virus with a specific receptor on the cell surface is the first and foremost step in virus infection. In case of enveloped viruses, their interaction with the host cell receptor is mediated by viral encoded glycoproteins on its envelope, a host derived lipid bilayer. Since, virus entry is a multistep process, after receptor recognition, envelope proteins mediate internalisation of virus particles into the host cell. Envelope glycoproteins are the first proteins that the host immune system encounters upon infection. Thus, envelope proteins are important drug target with multiple strategies to inhibit entry of the virus into the host. Currently, there are very few drugs that function as envelope protein inhibitors which are approved for human use. Here, we reviewed different classes of envelope proteins of various viruses and emphasised the use of small molecules to inhibit fusion of envelope proteins. Based on the available information in the literature, envelope proteins can be important drug targets and small molecules inhibitors can serve as potential antiviral drugs to block viral infection at an initial stage.

Heat shock proteins as biomarkers of lung cancer.

Heat shock proteins are known to be associated with a wide variety of human cancers including lung cancer. Overexpression of these molecular chaperones is linked with tumor survival, metastasis and anticancer drug resistance. In recent years, heat shock proteins are gaining much importance in the field of cancer research owing to their potential to be key determinants of cell survival and apoptosis. Lung cancer is one of the most common cancers diagnosed worldwide and the association of heat shock proteins in lung cancer diagnosis, prognosis and as drug targets remains unresolved. The aim of this review is to draw the importance of heat shock protein members; Hsp27, Hsp70, Hsp90, Hsp60 and their diagnostic and prognostic implications in lung cancer. Based on the available literature heat shock proteins can serve as biomarkers and anticancer drug targets in the management of lung cancer patients.

Recombinant viruses with other anti-cancer therapeutics: a step towards advancement of oncolytic virotherapy.

Cancer as a disease is a multifaceted foe which sometimes succumbs to the prescribed treatment and sometimes develops resistance against various therapies. Conventional cancer therapies suffer from many limitations, the least of which is their specificity and systemic side effects. In a majority of cases, acquired mutations render the cancer cells resistant to therapy and lower the prognostic outcome. In the constant effort to devise a therapeutic moiety which can comprehensively eliminate cancer cells, oncolytic viruses provide an attractive avenue as they selectively infect and lyse cancer cells sparing normal cells from their effects. Viruses can be engineered for their host specificity and toxicity as a promising anti-cancer tool. As it is essential to devise a strategy to address all targets involved in cancer development and progression, the idea of using oncolytic viruses with enhanced anti-cancer activity through arming with foreign genes gained merit and is showing promising advent in clinical studies. The use of oncolytic viruses as an agent of combination therapy for cancer treatment also gained much attention in the recent past. This review focuses on the emerging role of oncolytic viruses as vital components of anti-cancer regimen presenting a new dimension in an ever-changing cancer therapy scenario.

Combination of Oncolytic Measles Virus Armed With BNiP3, a Pro-apoptotic Gene and Paclitaxel Induces Breast Cancer Cell Death.

Breast cancer is one of the major causes of cancer related mortality in women worldwide. Limitations of conventional anti-cancer therapies such as severe systemic side effects, narrow therapeutic index, non-specificity, and non-availability of drugs for all types of cancers has resulted in the development of various novel and targeted approaches. The use of viruses as oncolytic agents has gained momentum for the development of an efficient therapeutic platform. In this study, we have developed recombinant measles virus armed with BNiP3, a pro-apoptotic gene of human origin, as an oncolytic agent, and have demonstrated its ability to induce apoptosis in breast cancer cells in vitro. Studies have demonstrated the potential of using oncolytic viruses in combination with conventional therapies as an efficient anti-cancer regimen. We also have explored the synergistic potential of this virus in combination with paclitaxel, and a hydrazone derivative, H2 compound as an anti-cancer agent. MCF-7 and MDA-MB-231, human breast cancer cell lines were used for in vitro studies to evaluate toxic effects of armed virus, rMV-BNiP3 both as a standalone therapy and in combination with paclitaxel or H2 compound, a hydrazone derivative. Generation of armed virus was confirmed by detecting the viral transcript and protein expression, while its oncolytic potential by cell viability assays. Induction of apoptosis was demonstrated by fluorescence based caspase 3 activity and flow cytometry based Annexin V/PI staining. In the current study we have demonstrated the successful generation of an oncolytic measles virus armed with BNiP3 (rMV-BNiP3) and the induction of toxic effects in rMV-BNiP3 infected cells with a curious bias toward MDA-MB-231 cells as compared to MCF-7. Infection of breast cancer cells with rMV-BNiP3 caused induction of cell death, but the combination of rMV-BNiP3 with sub-lethal doses of both paclitaxel and H2 lowered the overall viability of cancer cells. As triple negative breast tumors are highly aggressive and resistant subtype of breast cancer with poor prognosis, comparative sensitivity of MDA-MB-231 cells toward this virus may potentially be used to develop a targeted therapy against triple negative breast cancer.

Interaction of Host Nucleolin with Influenza A Virus Nucleoprotein in the Early Phase of Infection Limits the Late Viral Gene Expression.

Influenza A virus nucleoprotein, is a multifunctional RNA-binding protein, encoded by segment-5 of the negative sense RNA genome. It serves as a key connector between the virus and the host during virus replication. It continuously shuttles between the cytoplasm and the nucleus interacting with various host cellular factors. In the current study, host proteins interacting with nucleoprotein of Influenza A virus of H1N1 2009 pandemic strain were identified by co-immunoprecipitation studies followed by MALDI-TOF/MS analysis. Here we report the host nucleolin, a major RNA-binding protein of the nucleolus as a novel interacting partner to influenza A virus nucleoprotein. We thus, explored the implications of this interaction in virus life cycle and our studies have shown that these two proteins interact early during infection in the cytoplasm of infected cells. Depletion of nucleolin in A549 cells by siRNA targeting endogenous nucleolin followed by influenza A virus infection, disrupted its interaction with viral nucleoprotein, resulting in increased expression of gene transcripts encoding late viral proteins; matrix (M1) and hemagglutinin (HA) in infected cells. On the contrary, over expression of nucleolin in cells transiently transfected with pEGFP-NCL construct followed by virus infection significantly reduced the late viral gene transcripts, and consequently the viral titer. Altered expression of late viral genes and titers following manipulation of host cellular nucleolin, proposes the functional importance of its interaction with nucleoprotein during influenza A virus infection.

Synthesis and biological evaluation of 4-(2-(dimethylamino)ethoxy)benzohydrazide derivatives as inhibitors of Entamoeba histolyica.

In the quest for potent antiamoebic agents, a series of hydrazone hybrids (H1H30) have been designed and sequentially synthesized. The dimethylaminoethoxy and hydrazone entities incorporated into one molecule proved to be more persuasive and selective approach towards designing of antiamoebic agent. The synthesized compounds exhibited promising results against E. histolytica. The compound N'-(2-chlorobenzylidene)-4-(2-(dimethylamino) ethoxy)benzohydrazide was most impending among the series. Cytotoxicity profile showed better cell viability on lung cancer cell line (A549 cells) by MTT assay.

Proteomic analysis of HEK293 cells expressing non small cell lung carcinoma associated epidermal growth factor receptor variants reveals induction of heat shock response.

In view of the fact that certain non small cell lung carcinoma associated epidermal growth factor receptor mutations keep the receptor constitutively active, the downstream effectors of altered activity of mutant receptors are largely unknown. By 2D gel electrophoresis and MALDI-TOF/MS analysis, we showed that increased activity of EGFR mutants, L858R, L861Q and A871G induce heat shock proteins such as Hsp70, Hsp60, Hsp90B1, Hsp5a, Hsp71 and few transcriptional factors. Of which, Hsp70 was observed to be regulated more selectively to L861Q mutant. Our results suggest the possible role of heat shock proteins in lung tumor progression considering EGFR mutations.

Synthesis, characterization and antiamoebic activity of chalcones bearing N-substituted ethanamine tail.

A series of chalcones (4-21) possessing N-substituted ethanamine were synthesized by the aldol condensation reaction of 1-(4-(2-substituted ethoxy)phenyl)ethanones with different aldehydes preceded by the reaction of 2-chloro N-substituted ethanamine hydrochloride and 4-hydroxy acetophenone. The structure of all the synthesized compounds was elucidated by various spectral and X-ray diffraction studies. The compounds were screened against HM1: IMSS strain of Entamoeba histolytica and cytotoxicity was performed on A549 (non-small cell lung cancer cell line) cells by MTT assay. Out of eighteen compounds twelve showed better activity then the standard drug metronidazole. The compound 9, 14 and 19 showed good cell viability, hence were least toxic.

Corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/Akt activation.

Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3beta and nuclear translocation of the p65 subunit of NF-kappaB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.

Vitronectin: a possible determinant of adenovirus type 19 tropism for human corneal epithelium.

PURPOSE: Adenoviruses typically demonstrate specific tissue tropisms, as in the association of Ad19 with epidemic keratoconjunctivitis. We sought to determine factors that might influence the apparent tropism of Ad19 for the cornea. DESIGN: Laboratory investigation. METHODS: Adenovirus serotypes Ad2, 5, 9, 10, 11, 13, and 19 were compared for their capacity to replicate in human corneal epithelial cells (HCECs) in culture. Organotypically cultured human corneas were infected with Ad19 or Ad2, and viral titers were compared after 7 days. Replication of both viruses was compared in HCECs cultured on various extracellular matrices. Western blot analysis and immunohistochemistry were applied to human donor corneas and HCECs. RESULTS: One week after infection of HCEC monolayer cultures, Ad2 titers were significantly higher than any of the other viruses tested (P <.05). In organotypic corneal cultures, Ad19 titers were significantly higher than Ad2 (P = .0003). Ad2 replication in HCECs equaled or exceeded that of Ad19 on all extracellular matrices except vitronectin, where Ad2 replication was reduced and Ad19 replication enhanced (P <.0001). Vitronectin was detected by immunohistochemistry within the corneal epithelial basement membranes of human donor corneas. Increased alpha(v) integrin expression and greater tyrosine kinase phosphorylation in HCECs cultured on vitronectin were demonstrated by Western blot analysis. CONCLUSIONS: In vitro, vitronectin enhances growth of Ad19, possibly by up-regulation of receptor alpha(v) integrins and increased activity of tyrosine kinases necessary for adenoviral internalization. We hypothesize that differential tissue tropisms for adenoviruses may derive in part from tissue-specific extracellular matrix expression.

Corneal IL-8 expression following adenovirus infection is mediated by c-Src activation in human corneal fibroblasts.

Emerging evidence indicates that intracellular signaling cascades mediate entry of pathogenic adenoviruses into target host cells as well as some of the undesirable inflammatory responses to adenoviral gene vectors. We found that Ad19 infection of cultured human corneal fibroblasts induced IL-8 gene transcription independently of IL-1beta, TNF-alpha, and viral gene expression, suggesting that intracellular signaling events might mediate early inflammatory events in adenovirus keratitis. Heat but not UV light inactivation of the virus abrogated the effect of infection on IL-8 mRNA and protein levels, consistent with a viral binding-mediated mechanism. The tyrosine kinase inhibitor herbimycin blocked Ad19-induced IL-8 expression. Western blot analysis revealed tyrosine phosphorylation of the functionally related kinases c-Src and extracellular signal-regulated kinase (ERK) 1/2 in corneal fibroblasts within 15 min after infection. Respective inhibitors of these kinases, PP2 and PD98059, also blocked Ad19-induced IL-8 mRNA and protein expression. Application of inhibitors to Src and ERK kinase assays suggested an upstream relationship of c-Src to ERK. Finally, DNA microarray studies performed 1 h after Ad19 or mock infection of corneal fibroblasts in the presence or absence of the Src-specific inhibitor PP2 confirmed a relationship between c-Src and IL-8 expression in Ad19-infected corneal cells. c-Src may act as a global regulator of early proinflammatory host responses to Ad19 infection of the human cornea.

Genetic variability among Group A and B respiratory syncytial virus isolates from a large referral hospital in New Delhi, India.

Respiratory syncytial virus (RSV) is an important childhood pathogen of acute lower respiratory infections in developed and developing countries. The molecular epidemiology of RSV in India is largely unknown. The present study was undertaken to standardize and evaluate reverse transcription-PCR (RT-PCR) for the rapid and simultaneous detection of RSV groups A and B in clinical samples and to study intragroup genetic variability. RT-PCR was evaluated by comparing the results of seminested RT-PCR with centrifugation-enhanced cultures on 200 nasopharyngeal aspirates from children with acute lower respiratory infections. RSV was isolated in 34 nasopharyngeal aspirates by centrifugation-enhanced cultures and identified in 45 samples by RT-PCR. In 15 samples RSV was identified by seminested RT-PCR alone and in four by centrifugation-enhanced cultures alone. Of the 45 samples positive for RSV by nested PCR, 15 belonged to group A, 29 to group B, and one sample suggested a mixed infection. Group B RSV predominated in both years of the 2-year study. Genetic variability within RSV groups was studied by restriction fragment analysis of 35 PCR products. Among both group A and group B RSV, two different composite patterns were observed. Thus, RSV was found to be a major pathogen of acute lower respiratory tract infections in India, as it was detected in 24.5% of children by RT-PCR. RT-PCR provides a sensitive method for detection and typing of RSV group A and B viruses in clinical samples as well as a means to study intragroup variations. However, a higher sensitivity of detection of RSV in clinical samples can be obtained by its combination with additional techniques, such as virus cultivation.