The matrix (M) protein of newcastle disease virus binds to human bax through its BH3 domain

The underlying mechanisms by which Newcastle disease virus (NDV) kills cancer cells are still unclear. Recent discoveries have shown that many viruses contain Bcl-2 homology-like domains which enabled their interaction with Bcl-2 family members, and thereby accounting for their virulence and pathogenicity. Alignment of the protein sequences of Malaysian strain of NDV, known as AF2240, with those from members of the human Bcl-2 family showed many similar regions; most notably we found that its matrix (AF2240-M) protein, large (AF2240-L) protein and fusion (AF2240-F) protein all contain BH3-like regions. In addition, there are BH1-like domains in these proteins, where AF2240-F and Mcl-1 share 55% identity within this region. To further investigate our hypothesis that the presence of the BH3-like domains in these proteins may convey cytotoxicity, AF2240-M and AF2240-F genes were cloned into pFLAG and pEGFP.N2 vectors and transfected into HeLa cells. The expression of these constructs promoted cell death. As shown by flow cytometry, AF2240-M protein with deleted BH3-like region showed five-fold decrease in apoptosis. Moreover, the construct containing the N-terminal of AF2240-M showed nearly the same cell death rate as to that of the full-length protein, strongly suggesting that the BH3-like domain within this protein participates in promoting cell death. Moreover, AF2240-M transfection promoted Bax redistribution to mitochondria. Therefore, to determine whether there is any direct interaction between NDV viral proteins with some members of the Bcl-2 family, various constructs were co-transfected into HeLa cells. Co-immunoprecipitation trials showed that the AF2240-M indeed directly interacted with Bax protein via its BH3-domain, as the mutant proteins failed to interact with Bax. AF2240-F failed to interact with any of the tested proteins, although Bcl-XL slowed down the rate of cell death caused by this construct by nearly five-fold. In a parallel experiment, the level of expression of endogenous Bax and Bcl-2 after infection of HeLa cells with NDV was assessed by qRT-PCR, but no statistically significant change was observed. Consequently, the Bax/Bcl-2 ratio at the mRNA level did not alter. Overall, our study has shed additional light into the mechanisms by which NDV induces apoptosis

Newcastle Disease Virus Infection Promoted Bax-Mediated Apoptosis Assisted by the BH3 Domain of Matrix Protein

Newcastle disease virus (NDV) is a highly contagious avian paramyxovirus which leads to substantial losses in the poultry industry. NDV has gained widespread attention for its ability to selectively kill human cancer cells by apoptosis, and thus, it has been used as a cancer viro-therapeutic agent in many clinical trials. However, the exact mechanisms by which NDV induces apoptosis and kills cancer cells are still not entirely understood. Moreover, the precise signaling pathways leading to mitochondrial dysfunction are not entirely studied. In this study, the overall objective was to investigate if the intrinsic pathway is activated following NDV induced Apoptosis. Moreover, involvement of the Bcl-2 family of proteins needed to be comprehensively studied. First, the apoptotic effect of the Malaysian velogenic NDV strain AF2240 on HeLa and HT29 cancer cells was examined by DNA fragmentation and AO/PI staining. Immunoprecipitation and immunofluorescence experiments showed that NDV infection leads to a conformational change in the Bax protein and exposure of its N-terminal. Fractionation experiments showed that this change is associated with subcellular redistribution of Bax from the cytoplasm onto the mitochondria surface and subsequent release of cytochrome c. Densitometric and real-time PCR analyses revealed that the total levels of Bcl-2 and Bax proteins and mRNAs were not affected by NDV treatment, challenging previous findings that the ratio of Bax/Bcl-2 determines the fate of a cell during apoptosis. Alignment of all the protein sequences of AF2240, with those from members of the human Bcl-2 family revealed many identical regions; most notably the matrix (AF2240- M) protein, large (AF2240-L) protein and fusion (AF2240-F) proteins all contained BH3-like regions in their sequences with many shared conserved residues. To further investigate the hypothesis that the existence of the BH3-like domains in NDV viral proteins are cytotoxic, the AF2240-M and AF2240-F genes were cloned into pFLAG and pEGFP.N2 vectors and transfected into HeLa cells. The expressed proteins of these constructs promoted cell death. A mutant AF2240-M-BH3 which lacked the entire BH3 domain was then generated. Transfection of pFLAG-M-BH3 into HeLa cells showed a nearly five-fold decrease in cellular death. On the other hand, another construct which translated only 40 a.a. of the N-terminal of AF2240-M showed the same cell death rate as to that of the full-length protein, strongly suggesting that the BH3-like domain within this protein promotes apoptosis. Co-transfection and coimmunoprecipitation analyses to determine whether there is any direct interaction between AF2240-M or AF2240-F proteins with some members of the Bcl-2 family showed that the AF2240-M directly interacted with Bax protein via its BH3-domain, as the mutant proteins failed to interact with Bax. Overall, this study shows NDV contains homologs to human Bcl-2 family members that induce apoptosis. Moreover, these findings expand our understanding of how oncolytic strains of NDV kill cancer cells and suggest of a potential link between these proteins that may have occurred during early evolution

Rescue of recombinant Newcastle disease virus : current cloning strategies and RNA polymerase provision systems

Since the first rescue of a recombinant Newcastle disease virus (rNDV) in the late 1990s, many more rNDVs have been rescued by researchers around the world. Regardless of methodology, the main principle behind rescue of the virus has remained the same, i.e., the formation of a functional replication complex by simultaneously providing the full-length viral RNA and the viral NP, P and L proteins. However, different strategies have been reported for the insertion of the full-length genome into a suitable transcription vector, which remains the most challenging step of the rescue. Moreover, several systems have been published for provision of the DNA-dependent RNA polymerase, which is needed for transcription of viral RNA (vRNA) from the transfected plasmid DNA. The aim of this article is to consolidate all of the current cDNA assembly strategies and transcription systems used in rescue of rNDV in order to attain a better understanding of the advantages and disadvantages of each approach

Rescue of recombinant Newcastle disease virus : a short history of how it all started

Reverse genetics of viruses has come a long way, and many recombinant viruses have been generated since the first successful “rescues” were reported in the late 1970s. Recombinant Newcastle disease virus (rNDV), a non-segmented negative-sense RNA virus (NSNSV), was first rescued in 1999 using a reverse genetics approach similar to that reported for other recombinant viruses of the order Mononegavirales a few years before. The route from an original NDV isolate to the generation of its recombinant counterpart requires many steps that have to be sequentially and carefully completed. Background knowledge of each of these steps is essential because it allows one to make the best choices for fulfilling the specific requirements of the final recombinant virus. We have previously reviewed the latest strategies in cloning the NDV full-length cDNA into transcription vectors and the use of different RNA polymerase systems for the generation of viral RNA from plasmid DNA. In this article, we review a number of discoveries on the mechanism of transcription and replication of NDV, including a brief history behind the discovery of its RNP complex. This includes the generation of artificial and functional RNP constructs, in combination with the smart use of available knowledge and technologies that ultimately resulted in rescue of the first rNDV.</p

Recovery of a Malaysian recombinant Newcastle disease virus strain

Newcastle disease virus strain AF2240 is a viscerotropic velogenic strain that is used as a vaccine challenge virus in Malaysia. The identification of the full length genome will be a crucial platform for further studies of this isolate. In this study, we fully sequenced the genome of a derivative of this strain named AF2240-I. The 15,192 nt long genome contains a 55-nt leader sequence at the 3’ whereas the trailer region consists of 114-nt at the 5’. The intergenic sequences between the NP-P, P-M, M-F, F-HN and HN-L genes comprise 1, 1, 1, 31, and 47 nt respectively. The acknowledged cleavage site of fusion protein showed amino acid sequence of 112-R-R-Q-K-R-F-117 which corresponds to those of virulent NDV strains. Phylogenetic analysis of the whole virus genome showed that the strain AF2240-I belongs to genotype VIII and is more closely related to velogenic strains QH1, QH4, Fontana, Largo and Italien than to other strains of NDV. Differences were noticed in the HN and M gene between AF2240 and its derivative AF2240-I. This is the first report of a complete genome sequence of an NDV strain isolated in Malaysia

Characterization of Malaysian velogenic NDV strain AF2240-I genomic sequence: a comparative study

Newcastle disease virus (NDV) strain AF2240 is a viscerotropic velogenic strain that is used as a vaccine challenge virus in Malaysia. The identification of the full length genome will be a crucial platform for further studies of this isolate. In this study, we fully sequenced the genome of a derivative of this strain named AF2240-I. The 15,192 nt long genome contains a 55-nt leader sequence at the 3′ whereas the trailer region consists of 114 nt at the 5′. The intergenic sequences between the NP-P, P-M, M-F, F-HN, and HN-L genes comprise 1, 1, 1, 31, and 47 nt, respectively. The acknowledged cleavage site of fusion protein showed amino acid sequence of 112-R-R-Q-K-R-F-117, which corresponds to those of virulent NDV strains. Phylogenetic analysis of the whole virus genome shows that the strain AF2240-I belongs to genotype VIII and is more closely related to velogenic strains QH1, QH4, Fontana, Largo, and Italienas compared to other strains of NDV. Differences are noticed in the hemagglutinin-neuraminidase (HN) and matrix (M) gene between AF2240 and its derivative AF2240-I. This is the first report of a complete genome sequence of an NDV strain isolated in Malaysia

Rapid Generation of a Recombinant Genotype VIII Newcastle Disease Virus (NDV) Using Full-Length Synthetic cDNA

Rescue of (−)ssRNA viruses involves the sequential assembly and cloning of the full-length cDNA, which is often a challenging and time-consuming process. The objective of this study was to develop a novel method to rapidly clone the full-length cDNA of a very virulent NDV by only one assembly step. A completely synthetic 15 kb cDNA of a Malaysian genotype VIII NDV known as strain AF2240-I with additional flanking BsmBI sites was synthesised. However, to completely follow the rule-of-six, the additional G residues that are traditionally added after the T7 promoter transcription initiation site were not synthesised. The synthetic fragment was then cloned into low-copy number transcription vector pOLTV5-phiX between the T7 promoter and HDV Rz sequences through digestion with BbsI. The construct was co-transfected with helper plasmids into BSRT7/5 cells. A recombinant NDV called rAF was successfully rescued using transfection supernatant harvested as early as 16 h post-transfection. Virus from each passage showed an intracerebral pathogenicity index (ICPI) and a mean death time (MDT) similar to the parent strain AF2240-I. Moreover, rAF possessed an introduced mutation which was maintained for several passages. The entire rescue using the one-step assembly procedure was completed within a few weeks, which is extremely fast compared to previously used methods.</p

Virotherapy: current trends and future prospects for treatment of colon and rectal malignancies

Colorectal cancer (CRC) is one of the most common malignancies. In recent decades, early diagnosis and conventional therapies have resulted in a significant reduction in mortality. However, late stage metastatic disease still has very limited effective treatment options. There is a growing interest in using viruses to help target therapies to tumour sites. In recent years the evolution of immunotherapy has emphasised the importance of directing the immune system to eliminate tumour cells; we aim to give a state-of-the-art over-view of the diverse viruses that have been investigated as potential oncolytic agents for the treatment of CRC

Nutraceuticals: Transformation of Conventional Foods into Health Promoters/Disease Preventers and Safety Considerations

Nutraceuticals are essential food constituents that provide nutritional benefits as well as medicinal effects. The benefits of these foods are due to the presence of active compounds such as carotenoids, collagen hydrolysate, and dietary fibers. Nutraceuticals have been found to positively affect cardiovascular and immune system health and have a role in infection and cancer prevention. Nutraceuticals can be categorized into different classes based on their nature and mode of action. In this review, different classifications of nutraceuticals and their potential therapeutic activity, such as anti-cancer, antioxidant, anti-inflammatory and anti-lipid activity in disease will be reviewed. Moreover, the different mechanisms of action of these products, applications, and safety upon consumers including current trends and future prospect of nutraceuticals will be included