Structural Analysis of a Peptide (CTLTTKLYC) that Interacts with Newcastle Disease Virus

A peptide with the sequence Cys-Thr-Leu-Thr-Thr-Lys-Leu-Tyr-Cys (CTLTTKLYC) has previously been identified to inhibit the propagation of Newcastle disease virus (NDV) in embryonated chicken eggs and tissue culture. It has two different dissociation constants (&"I), in which the first constant can be used as a determinant to classifjr NDV strains into two groups: the velogenic strains in the first group, whereas the mesogenic and lentogenic strains are in the second group. The peptide, C 1T 2L3 P T5K 6 L7 Y8 C9 ,d isplayed on the pIII protein of a filamentous M13 phage was mutated by oligonucleotide-directed mutagenesis in order to identify the amino acid residues involved in the interactions with NDV. Mutations of Cys at first position (c')a nd Lys at the sixth position of the peptide ( K ~t)o Ala (A), which produced mutants C'A and K~Ad,i d not affect the binding between the peptide and the virus significantly, but substitution of Tyr at eighth position (Y8) alone with Ala (A) dramatically reduced the interaction. This suggests that y8 couid play an important role in the peptide-virus interaction. Double mutations were carried out on K~ and y8 to produce mutants K~A-Y~AK,~ R-Y~AK,~ A-Y'F, and K ~ - Y ~ Fto, determine whether the mutated amino acids could improve the binding capability. However, the mutations did not improve the binding capability significantly. Fmoc-solid phase peptide synthesis was employed to synthesize the peptide, CTLTTKLYC. Crude peptide was purified with HPLC and analysed with a mass spectrometer. The secondary structure of the peptide was analysed with circular dichroism (CD) and the three dimensional conformation of the peptide was determined by nuclear magnetic resonance (NMR) and molecular modelling. A mixture conformation of p-turn and P-sheet (intermolecular interaction) was observed for the linear peptide by using CD. However, the three-dimensional structure of the linear peptide could not be arrived due to the mixture of conformation which made the sequence assignment of NMR extremely difficult. On the other hand, the disulfide-constrained cyclic peptide, which has a more rigid structure, exhibited only a P-turn structure. Two models were obtained: one of it consists of a p-turn and a distorted p-turn, while the other structure is an extended structure

V protein, the virulence factor across the family Paramyxoviridae: a review

Paramyxoviridae is a family of viruses within the order Mononegavirales and comprises 14 genera; Metaavulavirus, Orthoavulavirus, Paraavulavirus, Synodonvirus, Ferlavirus, Aquaparamyxovirus, Henipavirus, Morbillivirus, Respirovirus, Jeilongvirus, Narmovirus, Salemvirus, Pararubulavirus and Orthorubulavirus. The members within this family are negative and single-stranded RNA viruses including human and animal pathogens such as measles virus (MeV), Nipah virus (NiV), mumps virus (MuV), Sendai virus (SeV) and Newcastle disease virus (NDV). The V protein is conserved within the family and plays an essential role in viral pathogenicity. Although V proteins of many paramyxoviruses are interferon-antagonists which counteract with the host’s innate immunity, there are still differences in the mode of action of the V protein between different genera or species within the same genera. The strategies to circumvent the host interferon (IFN) pathway can be divided into three general mechanisms; degradation of signal transducers and activators of transcription (STAT) protein, inhibition of phosphorylation of the transcription factor and, inhibition of translocation of STAT proteins into the nucleus. As a result, inhibition of IFN signalling and production promotes viral replication in the host cells. This review highlights the mechanism of the paramyxoviral V protein in evading the host IFN system

The effect of storage on the quality attributes of ultraviolet-irradiated and thermally pasteurised pineapple juices

The effect of storage time on the quality of ultraviolet-irradiated and thermally pasteurised pineapple juice was evaluated. The juices were irradiated with ultraviolet light (UV-C) at wavelength 254 nm (53.42 mJ/cm2, 4.918 s), thermally pasteurised at 800C for 10 minutes and stored at 40C for 13 weeks. There were significant changes in the total soluble solids, pH, titratable acidity and turbidity of UV-irradiated juice during storage, whereas for the same quality attributes of thermally pasteurised juice remained stable throughout the storage time. There were no significant changes in total phenolics for both treatments throughout the storage period. Other quality parameters (ascorbic acid, colour L, hue angle and chroma) were significantly affected by the storage time. Regarding the microbiological analysis, the total plate counts and yeast and mould counts of the UV-irradiated juice increased gradually throughout the 13 weeks of storage while these parameters remained unchanged in the thermally pasteurised juice with almost no microorganism growth. UV-irradiated pineapple juice preserved better quality attributes (TSS, pH, titratable acidity, ascorbic acid, turbidity, total phenolic, L (lightness), hue angle and chroma) than the thermal pasteurised juice during the storage time. Hence, UV irradiation has great potential as an alternative technology to thermal pasteurisation in producing products of high nutritive values

Lactococcus lactis: LAB model organism for bacteria-mediated therapeutic strategies

Lactococcus lactis is a well-characterized, food-grade lactic acid bacterium (LAB) with generally recognized as safe (GRAS) status. Better understanding of this bacterium at a molecular level has led to the development of unprecedented genetic tools that enable the expression of heterologous proteins. Subsequently, the ability of L. lactis to express and deliver these proteins to eukaryotic hosts presents a promising approach to achieve potent treatments for various diseases. Here, we have reviewed the characteristics of L. lactis and the expression systems established for this LAB model organism. We also described the experimental applications of L. lactis in disease therapy, especially its role as a vector in vaccination strategies

Newcastle disease virus strain AF2240 as an oncolytic virus: a review

The discovery of tumour selective virus-mediated apoptosis marked the birth of an alternative cancer treatment in the form of oncolytic viruses. Even though, its oncolytic efficiency was demonstrated more than 50 years ago, safety concerns which resulted from mild to lethal side effects hampered the progress of oncolytic virus research. Since the classical oncolytic virus studies rely heavily on its natural oncolytic ability, virus manipulation was limited, thereby, restricted efforts to improve its safety. In order to circumvent such restriction, experiments involving non-human viruses such as the avian Newcastle disease virus (NDV) was conducted using cultured cells, animal models and human subjects. The corresponding reports on its significant tumour cytotoxicity along with impressive safety profile initiated immense research interest in the field of oncolytic NDV. The varying degree of oncolytic efficiency and virulency among NDV strains encouraged researchers from all around the world to experiment with their respective local NDV isolates in order to develop an oncolytic virus with desirable characteristics. Such desirable features include high tumour-killing ability, selectivity and low systemic cytotoxicity. The Malaysian field outbreak isolate, NDV strain AF2240, also currently, receives significant research attention. Apart from its high cytotoxicity against tumour cells, this strain also provided fundamental insight into NDV-mediated apoptosis mechanism which involves Bax protein recruitment as well as death receptor engagement. Studies on its ability to selectively induce apoptosis in tumour cells also resulted in a proposed p38 MAPK/NF-κB/IκBα pathway. The immunogenicity of AF2240 was also investigated through PBMC stimulation and macrophage infection. In addition, the enhanced oncolytic ability of this strain under hypoxic condition signifies its dynamic tumour tropism. This review is aimed to introduce and discuss the aforementioned details of the oncolytic AF2240 strain along with its current challenges which outlines the future research direction of this virus

In-vitro and in-silico evaluations of heterocyclic-containing diarylpentanoids as Bcl-2 inhibitors against LoVo colorectal cancer cells

In the present study, we investigated the in-vitro anti-cancer potential of six diarylpentanoids against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, SW620, LoVo, HT29, NCI-H508, RKO, and LS411N cells. Structure-activity relationship study suggested that the insertions of tetrahydro-4H-thiopyran-4-one and brominated phenyl moieties are essential for better cytotoxicity. Among the evaluated analogs, 2e has been identified as the lead compound due to its low IC50 values of approximately 1 µM across all cancer cell lines and high chemotherapeutic index of 7.1. Anti-proliferative studies on LoVo cells showed that 2e could inhibit cell proliferation and colony formations by inducing G2/M cell cycle arrest. Subsequent cell apoptosis assay confirmed that 2e is a Bcl-2 inhibitor that could induce intrinsic cell apoptosis by creating a cellular redox imbalance through its direct inhibition on the Bcl-2 protein. Further molecular docking studies revealed that the bromophenyl moieties of 2e could interact with the Bcl-2 surface pocket through hydrophobic interaction, while the tetrahydro-4H-thiopyran-4-one fragment could form additional Pi-sulfur and Pi-alkyl interactions in the same binding site. In all, the present results suggest that 2e could be a potent lead that deserves further modification and investigation in the development of a new Bcl-2 inhibitor

Surface display of glycosylated Tyrosinase related protein-2 (TRP-2) tumour antigen on Lactococcus lactis

Background:The exploitation of the surface display system of food and commensal lactic acid bacteria (LAB) for bacterial, viral, or protozoan antigen delivery has received strong interest recently. The Generally Regarded as Safe (GRAS) status of the Lactococcus lactis coupled with a non-recombinant strategy of in-trans surface display, provide a safe platform for therapeutic drug and vaccine development. However, production of therapeutic proteins fused with cell-wall anchoring motifs is predominantly limited to prokaryotic expression systems. This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study, the glycosylated murine Tyrosinase related protein-2 (TRP-2) with the ability to anchor onto the L. lactis cell wall was produced in suspension adapted Chinese Hamster Ovary (CHO-S) cells by expressing TRP-2 fused with cell wall anchoring LysM motif (cA) at the C-terminus. Results: A total amount of 33 μg of partially purified TRP-2-cA from ~6.0 g in wet weight of CHO-S cells was purified by His-tag affinity chromatography. The purified TRP-2-cA protein was shown to be N-glycosylated and successfully anchored to the L. lactis cell wall. Conclusions: Thus cell surface presentation of glycosylated mammalian antigens may now permit development of novel and inexpensive vaccine platforms

Structural analysis of peptides that interact with Newcastle disease virus

Newcastle disease virus is a negativepolarity, single-stranded RNA virus (Lamb and Kolakofsky, 1996). It causes an economically important disease called Newcastle disease

An improved method for the rescue of of recombinant Newcastle disease virus

Reverse genetics has been used to generate recombinant Newcastle disease virus with enhanced immunogenic properties for vaccine development. The system, which involves co-transfecting the viral antigenomic plasmid with three helper plasmids into a T7 RNA polymerase-expressing cell to produce viral progenies, poses a great challenge. We have modified the standard transfection method to improve the transfection efficiency of the plasmids, resulting in a higher titer of virus progeny production. Two transfection reagents (i.e., lipofectamine and polyethylenimine) were used to compare the transfection efficiency of the four plasmids. The virus progenies produced were quantitated with flow cytometry analysis of the infectious virus unit. The modified transfection method increased the titer of virus progenies compared with that of the standard transfection method

Development of a T7 RNA polymerase expressing cell line using lentivirus vectors for the recovery of recombinant Newcastle disease virus

The development of a T7 RNA polymerase (T7 RNAP) expressing cell line i.e. BSR T7/5 cells marks an improvement of reverse genetics for the recovery of recombinant Newcastle disease virus (rNDV). BSR T7/5 is developed by transient transfection of plasmid encoding T7 RNAP gene for rNDV rescue. However, the gene expression decreases gradually over multiple passages and eventually hinders the rescue of rNDV. To address this issue, lentiviral vector was used to develop T7 RNAP-expressing HEK293-TA (HEK293-TA-Lv-T7) and SW620 (SW620-Lv-T7) cell lines, evidenced by the expression of T7 RNAP after subsequent 20 passages. rNDV was rescued successfully using HEK293-TA-Lv-T7 clones (R1D3, R1D8, R5B9) and SW620-Lv-T7 clones (R1C11, R3C5) by reverse transfection, yielding comparable virus rescue efficiency and virus titres to that of BSR T7/5. This study provides new tools for rNDV rescue and insights into cell line development and virology by reverse genetics