Modulation of programmed cell death pathways by the hepatitis C virus

10.2741/3709Frontiers in Bioscience162608-1

A putative diacidic motif in the SARS-CoV ORF6 protein influences its subcellular localization and suppression of expression of co-transfected expression constructs

<p>Abstract</p> <p>Background</p> <p>The ORF6 protein is one of the eight accessory proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV). Numerous properties of ORF6 have been documented and this study focuses on two of these, namely, its ability to suppress the expression of co-transfected expression constructs and its subcellular localization to vesicular structures.</p> <p>Results</p> <p>Using a transient transfection system, ORF6's ability to suppress the expression of co-transfected expression constructs was measured in a quantitative manner. While ORF6 does not have a global effect on protein synthesis, quantitative real-time PCR revealed that it down-regulated the mRNA level of the co-transfected myc-nsp8 gene. Furthermore, alanine substitution of a diacidic cluster motif (aa53-56) in the ORF6 gene caused a reduction in the suppression of expression of co-transfected myc-nsp8 gene. Our previous study revealed that ORF6 localized to vesicular structures in SARS-CoV infected Vero E6 cells. Here, ORF6 was observed to be localized to similar vesicular structures in Vero E6 cells which have been transiently transfected with a mammalian expression plasmid encoding for untagged ORF6. ORF6 showed partial colocalization with cellular proteins CD63 and Lamp1, suggesting that the vesicular structures may be a subpopulation of endosomal/lysosomal vesicles. The alanine substitution of the diacidic cluster motif also altered the subcellular localization of the ORF6 protein, indicating a potential relationship between the subcellular localization of the ORF6 protein and its ability to suppress the expression of co-transfected expression constructs.</p> <p>Conclusions</p> <p>By combining quantitative real-time PCR and transient transfection system, a simple and safe method is established to measure ORF6's ability to suppress the expression of co-transfected myc-nsp8. In addition, immunofluorescence analysis revealed that the subcellular localization of ORF6 when expressed on its own is similar to that observed in SARS-CoV infected cells. Through the use of these two assays, a putative diacidic motif in the ORF6 protein was found to influence its subcellular localization and ability to suppress the expression of co-transfected expression constructs.</p

Capital Punishment in Singapore: A Critical Analysis of State Justifications From 2004 to 2018

This article examines state justifications for capital punishment in Singapore. Singapore is a unique case study because capital punishment has largely been legitimised and justified by state officials. It illustrates how Singapore justifies capital punishment by analysing official discourse. Discussion will focus on the government’s narrative on capital punishment, which has been primarily directed against drug trafficking. Discussion will focus on Singapore’s death penalty regime and associated official discourse that seeks to justify state power to exercise such penalties, rather than the ethics and proportionality of capital punishment towards drug-related crimes. Critical analysis from a criminological perspective adds to the growing body of literature that seeks to conceptualise social and political phenomena in South-East Asia. &nbsp

Characterisation of human coronavirus-NL63 nucleocapsid protein

Coronavirus N is a multifunctional protein that plays an essential role in enhancing the efficiency of virus transcription and assembly. This manuscript reports the analysis of HCoV-NL63 N protein by comparing the amino acid sequences of coronavirus N-homologues. A ~50 kDa protein was expressed in both a mammalian cell and bacterial cell system that is similar in size to the predicted ~42.6 kDa HCoV-NL63 N protein. PSORTII identified two putative nuclear localisations signals and PONDR identified one disordered region in HCoV-NL63 N. The reported protein analysis serves as a prelude to laboratory analysis to understand the processing of HCoV-NL63 N.Web of Scienc

Characterisation of human coronavirus-NL63 nucleocapsid protein

Coronavirus N is a multifunctional protein that plays an essential role in enhancing the efficiency of virus transcription and assembly. This manuscript reports the analysis of HCoV-NL63 N protein by comparing the amino acid sequences of coronavirus N-homologues. A ~50 kDa protein was expressed in both a mammalian cell and bacterial cell system that is similar in size to the predicted ~42.6 kDa HCoV-NL63 N protein. PSORTII identified two putative nuclear localisations signals and PONDR identified one disordered region in HCoV-NL63 N. The reported protein analysis serves as a prelude to laboratory analysis to understand the processing of HCoV-NL63 N.Keywords: HCoV-NL63, nucleocapsid protein, protein analysis, nuclear localisation signals, protein ordered/disordered region

The p7 protein of the hepatitis C virus induces cell death differently from the influenza A virus viroporin M2

10.1016/j.virusres.2012.12.005Virus Research 1721-224-3

A Simple Methodology for Conversion of Mouse Monoclonal Antibody to Human-Mouse Chimeric Form

10.1155/2013/716961Clinical and Developmental Immunology2013Article number 716961,6 page

Understanding the T cell immune response in SARS coronavirus infection

10.1038/emi.2012.26Emerging Microbes and Infections1Article number e23, 6 page

A Novel Severe Acute Respiratory Syndrome Coronavirus Protein, U274, is transported to the Cell Surface and undergoes Endocytosis

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxx and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Coimmunoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.Web of Scienc

Memory T Cell Responses Targeting the SARS Coronavirus Persist up to 11 Years Post-Infection

10.1016/j.vaccine.2016.02.063Vaccine34172008–201