Discovery of 5-alkyl-2-pyrazol-oxazolidin-4-one derivatives as novel hepatitis B virus capsid assembly modulators.

The "magic methyl effect" strategy was used to design a series of 5-alkyl-2-pyrazol-oxazolidin-4-one derivatives as novel hepatitis B virus (HBV) capsid assembly modulators. Most of these compounds exhibited potent HBV inhibitory activities with low cytotoxicities in HepG2.2.15 cells. The most promising compounds 9d and 10b had single-digit nanomolar IC50 values with a high selectivity index. Compared with the lead compound (3.0%), they caused 15% and 18% decreases in HBe antigen secretion at 1.0 µM, respectively. In addition, compounds 9d and 10b possessed good pharmacokinetic profiles with oral bioavailability values of 56.1% and 48.9%, respectively. These results indicated that the two compounds were potential therapeutic agents for HBV infection.

Design of novel peptide inhibitors against the conserved bacterial transcription terminator, Rho.

The transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA remodeling, and so forth, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C termini. Expression of these peptides inhibited the growth of Escherichia coli and specifically inhibited Rho-dependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if eight to ten amino acids were deleted from their C termini. In silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA-binding site of the Rho hexamer near its subunit interfaces. In addition, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria and inhibited the activities of Rho proteins from Mycobacterium tuberculosis, Xanthomonas, Vibrio cholerae, and Salmonella enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the ∼42-kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel antimicrobial peptides.

Structure-based virtual screening workflow to identify antivirals targeting HIV-1 capsid.

We have identified novel HIV-1 capsid inhibitors targeting the PF74 binding site. Acting as the building block of the HIV-1 capsid core, the HIV-1 capsid protein plays an important role in the viral life cycle and is an attractive target for antiviral development. A structure-based virtual screening workflow for hit identification was employed, which includes docking 1.6 million commercially-available drug-like compounds from the ZINC database to the capsid dimer, followed by applying two absolute binding free energy (ABFE) filters on the 500 top-ranked molecules from docking. The first employs the Binding Energy Distribution Analysis Method (BEDAM) in implicit solvent. The top-ranked compounds are then refined using the Double Decoupling method in explicit solvent. Both docking and BEDAM refinement were carried out on the IBM World Community Grid as part of the FightAIDS@Home project. Using this virtual screening workflow, we identified 24 molecules with calculated binding free energies between - 6 and - 12 kcal/mol. We performed thermal shift assays on these molecules to examine their potential effects on the stability of HIV-1 capsid hexamer and found that two compounds, ZINC520357473 and ZINC4119064 increased the melting point of the latter by 14.8 °C and 33 °C, respectively. These results support the conclusion that the two ZINC compounds are primary hits targeting the capsid dimer interface. Our simulations also suggest that the two hit molecules may bind at the capsid dimer interface by occupying a new sub-pocket that has not been exploited by existing CA inhibitors. The possible causes for why other top-scored compounds suggested by ABFE filters failed to show measurable activity are discussed.

Membrane intercalation-enhanced photodynamic inactivation of bacteria by a metallacycle and TAT-decorated virus coat protein.

Antibiotic resistance has become one of the major threats to global health. Photodynamic inactivation (PDI) develops little antibiotic resistance; thus, it becomes a promising strategy in the control of bacterial infection. During a PDI process, light-induced reactive oxygen species (ROS) damage the membrane components, leading to the membrane rupture and bacteria death. Due to the short half-life and reaction radius of ROS, achieving the cell-membrane intercalation of photosensitizers is a key challenge for PDI of bacteria. In this work, a tetraphenylethylene-based discrete organoplatinum(II) metallacycle (1) acts as a photosensitizer with aggregation-induced emission. It self-assembles with a transacting activator of transduction (TAT) peptide-decorated virus coat protein (2) through electrostatic interactions. This assembly (3) exhibits both ROS generation and strong membrane-intercalating ability, resulting in significantly enhanced PDI efficiency against bacteria. By intercalating in the bacterial cell membrane or entering the bacteria, assembly 3 decreases the survival rate of gram-negative Escherichia coli to nearly zero and that of gram-positive Staphylococcus aureus to ∼30% upon light irradiation. This study has wide implications from the generation of multifunctional nanomaterials to the control of bacterial infection, especially for gram-negative bacteria.

Reovirus σ3 Protein Limits Interferon Expression and Cell Death Induction.

Induction of necroptosis by mammalian reovirus requires both type I interferon (IFN)-signaling and viral replication events that lead to production of progeny genomic double-stranded RNA (dsRNA). The reovirus outer capsid protein µ1 negatively regulates reovirus-induced necroptosis by limiting RNA synthesis. To determine if the outer capsid protein σ3, which interacts with µ1, also functions in regulating necroptosis, we used small interfering RNA (siRNA)-mediated knockdown. Similarly to what was observed in diminishment of µ1 expression, knockdown of newly synthesized σ3 enhances necroptosis. Knockdown of σ3 does not impact reovirus RNA synthesis. Instead, this increase in necroptosis following σ3 knockdown is accompanied by an increase in IFN production. Furthermore, ectopic expression of σ3 is sufficient to block IFN expression following infection. Surprisingly, the capacity of σ3 protein to bind dsRNA does not impact its capacity to diminish production of IFN. Consistent with this, infection with a virus harboring a mutation in the dsRNA binding domain of σ3 does not result in enhanced production of IFN or necroptosis. Together, these data suggest that σ3 limits the production of IFN to control innate immune signaling and necroptosis following infection through a mechanism that is independent of its dsRNA binding capacity.IMPORTANCE We use mammalian reovirus as a model to study how virus infection modulates innate immune signaling and cell death induction. Here, we sought to determine how viral factors regulate these processes. Our work highlights a previously unknown role for the reovirus outer capsid protein σ3 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires production of interferon. The σ3 protein limits the induction of necroptosis by preventing excessive production of interferon following infection.

Generation of a soluble and stable apoptin-EGF fusion protein, a targeted viral protein applicable for tumor therapy.

A promising candidate for tumor targeted toxins is the chicken anemia-derived protein apoptin that induces tumor-specific apoptosis. It was aimed to design a novel apoptin-based targeted toxin by genetic fusion of apoptin with the tumor-directed ligand epidermal growth factor (EGF) using Escherichia coli as expression host. However, apoptin is highly hydrophobic and tends to form insoluble aggregates. Therefore, three different apoptin-EGF variants were generated. The fusion protein hexa-histidine (His)-apoptin-EGF (HAE) was expressed in E. coli and purified under denaturing conditions due to inclusion bodies. The protein solubility was improved by maltose-binding protein (MBP) or glutathione S-transferase. The protein MBP-apoptin-EGFHis (MAEH) was found favorable as a targeted toxin regarding final yield (4-6 mg/L) and stability. MBP was enzymatically removed using clotting factor Xa, which resulted in low yield and poor separation. MAEH was tested on target and non-target cell lines. The targeted tumor cell line A431 showed significant toxicity with an IC50 of 69.55 nM upon incubation with MAEH while fibroblasts and target receptor-free cells remained unaffected. Here we designed a novel EGF receptor targeting drug with high yield, purity and stability.

Highly efficient cellular uptake of a cell-penetrating peptide (CPP) derived from the capsid protein of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is one of the smallest, nonenveloped, single-stranded DNA viruses. The PCV2 capsid protein (Cap) is the sole viral structural protein and main antigenic determinant. Previous sequence analysis has revealed that the N terminus of the PCV2 Cap contains a nuclear localization signal (NLS) enriched in positively charged residues. Here, we report that PCV2's NLS can function as a cell-penetrating peptide (CPP). We observed that this NLS can carry macromolecules, e.g. enhanced GFP (EGFP), into cells when they are fused to the NLS, indicating that it can function as a CPP, similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT). We also found that the first 17 residues of the NLS (NLS-A) have a key role in cellular uptake. In addition to entering cells via multiple endocytic processes, NLS-A was also rapidly internalized via direct translocation enabled by increased membrane permeability and was evenly distributed throughout cells when its concentration in cell cultures was ≥10 µm Of note, cellular NLS-A uptake was ∼10 times more efficient than that of HIV TAT. We inferred that the externalized NLS of the PCV2 Cap may accumulate to a high concentration (≥10 µm) at a local membrane area, increasing membrane permeability to facilitate viral entry into the cell to release its genome into a viral DNA reproduction center. We conclude that NLS-A has potential as a versatile vehicle for shuttling foreign molecules into cells, including pharmaceuticals for therapeutic interventions.

Knob protein enhances epithelial barrier integrity and attenuates airway inflammation.

BACKGROUND: Altered epithelial physical and functional barrier properties along with TH1/TH2 immune dysregulation are features of allergic asthma. Regulation of junction proteins to improve barrier function of airway epithelial cells has the potential for alleviation of allergic airway inflammation. OBJECTIVE: We sought to determine the immunomodulatory effect of knob protein of the adenoviral capsid on allergic asthma and to investigate its mechanism of action on airway epithelial junction proteins and barrier function. METHODS: Airway inflammation, including junction protein expression, was evaluated in allergen-challenged mice with and without treatment with knob. Human bronchial epithelial cells were exposed to knob, and its effects on expression of junction proteins and barrier integrity were determined. RESULTS: Administration of knob to allergen-challenged mice suppressed airway inflammation (eosinophilia, airway hyperresponsiveness, and IL-5 levels) and prevented allergen-induced loss of airway epithelial occludin and E-cadherin expression. Additionally, knob decreased expression of TH2-promoting inflammatory mediators, specifically IL-33, by murine lung epithelial cells. At a cellular level, treatment of human bronchial epithelial cells with knob activated c-Jun N-terminal kinase, increased expression of occludin and E-cadherin, and enhanced epithelial barrier integrity. CONCLUSION: Increased expression of junction proteins mediated by knob leading to enhanced epithelial barrier function might mitigate the allergen-induced airway inflammatory response, including asthma.

A Bacteriophage Capsid Protein Is an Inhibitor of a Conserved Transcription Terminator of Various Bacterial Pathogens.

Rho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits Escherichia coli Rho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, including Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium tuberculosis, Xanthomonas campestris, Xanthomonas oryzae, Corynebacterium glutamicum, Vibrio cholerae, Salmonella enterica, and Pseudomonas syringae The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pulldown assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis, M. bovis, X. campestris, and E. coli expressing S. enterica Rho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the "universal" inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCE Bacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of the E. coli transcription terminator Rho. Here we show that apart from antagonizing E. coli Rho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psu in vivo kills various pathogens, such as Mycobacterium and Xanthomonas species. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.

rApoptin induces apoptosis in human breast cancer cells via phosphorylation of Nur77 and Akt.

Breast cancer is the leading cause of cancer incidence and cancer-related mortality among women and is becoming a major public health problem around the world. The current study aims to investigate the possible role and mechanism of recombinant Apoptin (rApoptin), a potential anticancer candidate that minimally impacts normal cells, in the breast cancer cell proliferation and apoptosis in vitro and in vivo. We found that rApoptin could effectively inhibit the proliferation and apoptosis in MCF-7 and MDA-MB-231 cells in vitro, which was further confirmed by flow cytometry analysis. Apoptin partially inhibited MCF-7 cell xenograft tumor development in vivo. Furthermore, we found via western blot that rApoptin-induced apoptosis in MCF-7 and MDA-MB-231 cells was associated with the phosphorylation of Nur77 (p-Nur77) and Akt (p-Akt). In addition, compared with the control groups, rApoptin-treated tissues showed significantly higher expression of Bax and Cyt c while Bcl-2 expression was decreased by rApoptin treatment. Together, our results are the first to demonstrate that rApoptin was able to effectively induce breast cancer cell apoptosis both in vitro and in vivo and that this activity could be regulated by the phosphorylation of Nur77 and Akt and the mitochondrial pathway. Our findings highlight the potential application of rApoptin as a breast cancer treatment.

A truncated apoptin protein variant selectively kills cancer cells.

Apoptin is a nonstructural protein encoded by one of the three open reading frames of the chicken anemia virus genome. It has attracted a great deal of interest due to its ability to induce apoptosis in multiple transformed and malignant mammalian cell lines without affecting primary and non-transformed cells. However, the use of Apoptin as an anticancer drug is restricted by its strong tendency to aggregate. A number of methods to overcome this problem have been proposed, including transduction techniques to deliver the Apoptin gene into tumor cells, but all such methods have certain drawbacks. Here we describe that a truncated variant of Apoptin, lacking residues 1 to 43, is a soluble, non-aggregating protein that maintains most of the biological properties of wild-type Apoptin when transfected into cells. We show that the cytotoxic effect of this variant is also present when it is added exogenously to cancer cells, but not to normal cells. In addition to the interest this protein has attracted as a promising therapeutic strategy, it is also an excellent model to study the structural properties of Apoptin and how they relate to its mechanism of action.

Identification of proteins differentially expressed by Chlamydia trachomatis treated with chlamydiaphage capsid protein VP1 during intracellular growth.

Chlamydia trachomatis infection is one of the most prevalent sexually transmitted diseases. Our research pertains to the inhibitory effect and molecular mechanism of the chlamydiaphage capsid protein VP1 on the growth of Chlamydia trachomatis. In this research, the capsid protein VP1 of the guinea-pig conjunctivitis chlamydiaphage phiCPG1 was expressed, purified and identified, and then, it was applied to the cultivation of different serovars of Chlamydia trachomatis and Chlamydia psittaci. The inhibitory effect was observed in each serovar of Chlamydia trachomatis (D, E, F, G, H, I, K, and L2) and Chlamydia psittaci inoculated with VP1 protein. The inhibition affection of VP1 on the growth of Chlamydia trachomatis was caused by the changes of expressions of some related proteins including 36 proteins up-regulated and 81 proteins down-regulated in the development cycle of Ct through the label-free test, and the transcription levels of these proteins, including Hc1, pmpD, and MOMP, were confirmed by RT-PCR. It provides information that is essential for understanding the mechanism of chlamydiaphage capsid protein VP1 on chlamydia and a new direction for further clinical treatment of chlamydial infection.

Unexpected heat shock element binding ability and tumor-killing activity of the combinatorial function domain of apoptin.

Apoptin, derived from the chicken anemia virus, has been found to exert tumor-preferential apoptotic activity. It is a potential anticancer agent with direct clinical applications. However, if this viral protein were to be used as a new drug, it might also induce a strong immune response, causing toxic side effects. In a previous study, our group showed that TAT-apoptin downregulates the stress expression of heat shock protein 70 by competing with heat shock factor protein 1 in binding to the heat shock element (HSE) of the promoter region of heat shock protein 70, thus inducing specific apoptosis in HepG2 cells. In this study, we investigated the HSE-binding properties of the minimal functional region of apoptin. We showed that apoptin's nuclear localization signals 1 and nuclear localization signals 2 represented functional regions that could bind with HSE and that this binding capacity was increased by polymers formed through the introduction of a leucine-rich stretch. Our data also showed that truncated combinatorial apoptin peptide has greater tumor-specific cell-killing activity and could be a potential antitumor agent.

Immobilization of bacteriophage in wound-dressing nanostructure.

Opportunistic bacteria that cause life-threatening infections are still a central problem associated with a healthcare setting. Bacteriophage capsid immobilization on nanostructured polymers maximizes its tail exposure and looks promising in applications toward skin-infections as alternative to antibiotics standardly used. The main goal of this work was to investigate the covalent immobilization of vB_Pae_Kakheti25 bacteriophage capsid on polycaprolactone (PCL) nanofibers (non-woven textile), as a potential effective antimicrobial, laundry resistant and non-toxic dressing for biomedical use. Surface analyses showed that the immobilization of vB_Pae_Kakheti25 bacteriophage capsid on PCL nanofibres oriented bacteriophage tails to interact with bacteria. Furthermore, antimicrobial assays showed a very effective 6 log bacterial reduction, which was equivalent to 99.9999%, after immediate and 2 hours of contact, even following 25 washing cycles (due to covalent bond). The activity of PCL-vB_Pae_Kakheti25 against P. aeruginosa was immediate and its reduction was complete.

Molecular cloning, expression and characterization of 100K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome.

Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage.

Cross-serotype immunity induced by immunization with a conserved rhinovirus capsid protein.

Human rhinovirus (RV) infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2) capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.

A herpes simplex virus scaffold peptide that binds the portal vertex inhibits early steps in viral replication.

Previous experiments identified a 12-amino-acid (aa) peptide that was sufficient to interact with the herpes simplex virus 1 (HSV-1) portal protein and was necessary to incorporate the portal into capsids. In the present study, cells were treated at various times postinfection with peptides consisting of a portion of the Drosophila antennapedia protein, previously shown to enter cells efficiently, fused to either wild-type HSV-1 scaffold peptide (YPYYPGEARGAP) or a control peptide that contained changes at positions 4 and 5. These 4-tyrosine and 5-proline residues are highly conserved in herpesvirus scaffold proteins and were previously shown to be critical for the portal interaction. Treatment early in infection with subtoxic levels of wild-type peptide reduced viral infectivity by over 1,000-fold, while the mutant peptide had little effect on viral yields. In cells infected for 3 h in the presence of wild-type peptide, capsids were observed to transit to the nuclear rim normally, as viewed by fluorescence microscopy. However, observation by electron microscopy in thin sections revealed an aberrant and significant increase of DNA-containing capsids compared to infected cells treated with the mutant peptide. Early treatment with peptide also prevented formation of viral DNA replication compartments. These data suggest that the antiviral peptide stabilizes capsids early in infection, causing retention of DNA within them, and that this activity correlates with peptide binding to the portal protein. The data are consistent with the hypothesis that the portal vertex is the conduit through which DNA is ejected to initiate infection.

Apoptin selectively induces the apoptosis of tumor cells by suppressing the transcription of HSP70.

Apoptin is a nonstructural viral protein encoded by VP3 gene of chicken anemia virus, which could specially induce apoptosis of tumor cells. However, the mechanism of apoptin-induced apoptosis in tumor cells without any side effects in normal cells has not yet been well characterized. This study aimed to investigate the molecular mechanism underlying the selective antitumor effects of apoptin. HepG2 cells were treated with apoptin or transfected with apoptin expression vector. Heat shock protein 70 (HSP70) expression was examined by Western blot. The binding of apoptin to HSP70 promoter was detected by electrophoretic mobility shift assay, chromatin immunoprecipitation, and luciferase assay. The results showed that apoptin inhibited HSP70 expression in HepG2 cells and apoptin-induced apoptosis of HepG2 cells was dependent on the expression level of HSP70. Furthermore, apoptin promoted HSF1 trimer depolymerization and inhibited HSF1-mediated HSP70 transcription. In addition, apoptin competed with HSF1 to bind heat shock element in HSP70 promoter, leading to reduced HSP70 transcription. Both these mechanisms contribute to the suppression of HSP70 transcription and expression. Our findings provide the first evidence that apoptin induces tumor cell apoptosis by specifically downregulating the expression of HSP70, which helps explain the specific antitumor effects of apoptin.

Selective apoptosis induction in MCF-7 cell line by truncated minimal functional region of Apoptin.

BACKGROUND: Chicken Anemia Virus (CAV) VP3 protein (also known as Apoptin), a basic and proline-rich protein has a unique capability in inducing apoptosis in cancer cells but not in normal cells. Five truncated Apoptin proteins were analyzed to determine their selective ability to migrate into the nucleus of human breast adenocarcinoma MCF-7 cells for inducing apoptosis. METHODS: For identification of the minimal selective domain for apoptosis, the wild-type Apoptin gene had been reconstructed by PCR to generate segmental deletions at the N' terminal and linked with nuclear localization sites (NLS1 and NLS2). All the constructs were fused with maltose-binding protein gene and individually expressed by in vitro Rapid Translation System. Standardized dose of proteins were delivered into human breast adenocarcinoma MCF-7 cells and control human liver Chang cells by cytoplasmic microinjection, and subsequently observed for selective apoptosis effect. RESULTS: Three of the truncated Apoptin proteins with N-terminal deletions spanning amino acid 32-83 retained the cancer selective nature of wild-type Apoptin. The proteins were successfully translocated to the nucleus of MCF-7 cells initiating apoptosis, whereas non-toxic cytoplasmic retention was observed in normal Chang cells. Whilst these truncated proteins retained the tumour-specific death effector ability, the specificity for MCF-7 cells was lost in two other truncated proteins that harbor deletions at amino acid 1-31. The detection of apoptosing normal Chang cells and MCF-7 cells upon cytoplasmic microinjection of these proteins implicated a loss in Apoptin's signature targeting activity. CONCLUSIONS: Therefore, the critical stretch spanning amino acid 1-31 at the upstream of a known hydrophobic leucine-rich stretch (LRS) was strongly suggested as one of the prerequisite region in Apoptin for cancer targeting. Identification of this selective domain provides a platform for developing small targets to facilitating carrier-mediated-transport across cellular membrane, simultaneously promoting protein delivery for selective and effective breast cancer therapy.

MEK5/ERK5/mef2: a novel signaling pathway affected by hepatitis C virus non-enveloped capsid-like particles.

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The viral particle is composed of a capsid containing the genome, surrounded by E1 and E2 proteins, however different forms of viral particles have been observed including non-enveloped particles. Previous reports have proposed that hepatitis C non-enveloped capsid-like particles (HCVne) enter cells of hepatic origin via clathrin-mediated endocytosis, during which different signaling events occur. In this report we show that HCVne particles are capable of inducing the recently discovered ERK5 pathway, in a dose dependent way. The ERK5 pathway can be activated by growth factors and other extracellular signals. This specific activation occurs through a well characterized upstream kinase, MEK5, and is capable of inducing gene regulation of mef2. In contrast, when HCV core structural and NS5A non-structural proteins were expressed endogenously no activation of this pathway was detected. These cell signaling events could be of critical importance and might give clues for the elucidation of cellular manifestations associated with HCV infection.