scholarly journals Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice

2005 ◽  
Vol 86 (1) ◽  
pp. 211-215 ◽  
Author(s):  
Alexander N. Zakhartchouk ◽  
Sathiyanarayanan Viswanathan ◽  
James B. Mahony ◽  
Jack Gauldie ◽  
Lorne A. Babiuk
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Immune Responses ◽  
Nucleocapsid Protein ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
Cell Mediated Immunity ◽  
Aetiological Agent ◽  
N Protein ◽  
Adenovirus 5

Severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Thus, vaccination against SARS-CoV may represent an effective approach towards controlling SARS. The nucleocapsid (N) protein is thought to play a role in induction of cell-mediated immunity to SARS-CoV and thus it is important to characterize this protein. In the present study, an E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector (Ad5-N-V) expressing the SARS-CoV N protein was constructed. The N protein, expressed in vitro by Ad5-N-V, was of the expected molecular mass of 50 kDa and was phosphorylated. Vaccination of C57BL/6 mice with Ad5-N-V generated potent SARS-CoV-specific humoral and T cell-mediated immune responses. These results show that Ad5-N-V may potentially be used as a SARS-CoV vaccine.

scholarly journals The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein Is Phosphorylated and Localizes in the Cytoplasm by 14-3-3-Mediated Translocation

2005 ◽  
Vol 79 (17) ◽  
pp. 11476-11486 ◽  
Author(s):  
Milan Surjit ◽  
Ravinder Kumar ◽  
Rabi N. Mishra ◽  
Malireddy K. Reddy ◽  
Vincent T. K. Chow ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Protein Interactions ◽  
Nucleocapsid Protein ◽  
Glycogen Synthase ◽  
In Silico Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Pathways ◽  
N Protein ◽  
Mitogen Activated Protein

ABSTRACT The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3θ), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3θ we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.

scholarly journals Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

2021 ◽  
Author(s):  
Christine Roden ◽  
Yifan Dai ◽  
Ian Seim ◽  
Myungwoon Lee ◽  
Rachel Sealfon ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Structure ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Genome Packaging ◽  
Rna Motifs ◽  
Double Stranded Rna ◽  
N Protein ◽  
Binding Domains

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

scholarly journals QUANTITATIVE EFFECTS OF NUTRITIONAL ESSENTIAL AMINO ACID DEFICIENCY UPON IMMUNE RESPONSES TO TUMORS IN MICE

1973 ◽  
Vol 137 (1) ◽  
pp. 1-9 ◽  
Author(s):  
David G. Jose ◽  
Robert A. Good
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Immune Responses ◽  
Tumor Growth ◽  
Essential Amino Acid ◽  
Cell Mediated Immunity ◽  
Host Animal ◽  
Vitro Assay ◽  
Cytotoxic Cell

Mice were fed diets deficient in a single essential amino acid, and the primary immune responses to inoculation of allogenic tumor cells was measured by in vitro assay of cellular immunity. Moderate reduction of the amino acids phenylalanine-tyrosine, valine, threonine, methionine-cystine, isoleucine, and tryptophane in the diet produced profound depression of hemagglutinating and blocking antibody responses, although cytotoxic cell-mediated immunity remained intact. These diets had previously been shown to result in a selective depression of tumor growth in mice. Limitation of the amino acids arginine, histidine, and lysine in the diets gave rise to only slight depression of the immune responses. These diets had previously been shown to produce a proportional decrease in both tumor growth and host body weight. Moderate leucine restriction resulted in a paradoxical depression of cytotoxic cell-mediated immunity with little effect on serum blocking activity. Slight increases had previously been noted in the weight of tumors in mice fed leucine-restricted diets. Deficiency or imbalance of essential amino acids in the diet may produce profound depression of immune responses and apparent, marked changes in the immune resistance of the host animal to tumors.

Impaired In Vivo Immune Responses in Patients with Melancholia

1993 ◽  
Vol 162 (5) ◽  
pp. 651-657 ◽  
Author(s):  
Ian Hickie ◽  
Catherine Hickie ◽  
Andrew Lloyd ◽  
Derrick Silove ◽  
Denis Wakefield
Keyword(s):  
Immune Responses ◽  
Delayed Type Hypersensitivity ◽  
Cell Mediated Immunity ◽  
Melancholic Depression ◽  
Skin Responses ◽  
Severity Of Depression ◽  
Depressive Subtypes ◽  
Control Study

Previous attempts to establish a relationship between impaired cell-mediated immunity (CMI) and major mood disorders have been limited by a failure to explore the relevance of depressive subcategories or to assess CMI by in vivo methods. In this case-control study CMI was assessed in 57 patients with major depression (31 with melancholic, 26 with non-melancholic disorders), and in age- and sex-matched controls by both in vitro and in vivo immunological techniques. Compared with control subjects and patients with non-melancholic depression, patients with melancholia demonstrated reduced in vivo CMI as assessed by delayed-type hypersensitivity (DTH) skin responses. Although increasing age, severity of depression, hospital admission for treatment, and reported weight loss are correlates of melancholia, none of these factors alone, or in combination, accounted for the differences in DTH responses observed between the two depressive subtypes. These data suggest that impaired CMI in vivo may be limited to those with melancholic disorders. At this stage the factors which account for this effect are unclear.

scholarly journals The 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in Vero E6 cells

2005 ◽  
Vol 86 (7) ◽  
pp. 1921-1930 ◽  
Author(s):  
Patrick T. W. Law ◽  
Chi-Hang Wong ◽  
Thomas C. C. Au ◽  
Chi-Pang Chuck ◽  
Siu-Kai Kong ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Severe Acute Respiratory Syndrome ◽  
Viral Infection ◽  
Viral Protein ◽  
Host Response ◽  
Transmembrane Protein ◽  
Chromatin Condensation ◽  
Aetiological Agent ◽  
First Case

An outbreak of severe acute respiratory syndrome (SARS) occurred in China and the first case emerged in mid-November 2002. The aetiological agent of this disease was found to be a previously unknown coronavirus, SARS-associated coronavirus (SARS-CoV). The detailed pathology of SARS-CoV infection and the host response to the viral infection are still not known. The 3a gene encodes a non-structural viral protein, which is predicted to be a transmembrane protein. In this study, it was shown that the 3a protein was expressed in the lungs and intestinal tissues of SARS patients and that the protein localized to the endoplasmic reticulum in 3a-transfected monkey kidney Vero E6 cells. In vitro experiments of chromatin condensation and DNA fragmentation suggested that the 3a protein may trigger apoptosis. These data showed that overexpression of a single SARS-CoV protein can induce apoptosis in vitro.

scholarly journals Molecular and Biological Characterization of Human Monoclonal Antibodies Binding to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome Coronavirus

2005 ◽  
Vol 79 (3) ◽  
pp. 1635-1644 ◽  
Author(s):  
Edward N. van den Brink ◽  
Jan ter Meulen ◽  
Freek Cox ◽  
Mandy A. C. Jongeneelen ◽  
Alexandra Thijsse ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Receptor Binding ◽  
Vero Cells ◽  
Human Monoclonal Antibodies ◽  
Human Mabs ◽  
N Protein ◽  
Antibody Phage ◽  
Antibody Phage Display

ABSTRACT Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.

scholarly journals Evaluation of a Fiber-Modified Adenovirus Vector Vaccine against Foot-and-Mouth Disease in Cattle

2015 ◽  
Vol 23 (2) ◽  
pp. 125-136 ◽  
Author(s):  
Gisselle N. Medina ◽  
Nestor Montiel ◽  
Fayna Diaz-San Segundo ◽  
Diego Sturza ◽  
Elizabeth Ramirez-Medina ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Clinical Signs ◽  
Foot And Mouth Disease ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
Mouth Disease ◽  
Cell Mediated Immunity ◽  
Serum Samples ◽  
Vector Vaccine ◽  
Foot And Mouth

ABSTRACTNovel vaccination approaches against foot-and-mouth disease (FMD) include the use of replication-defective human adenovirus type 5 (Ad5) vectors that contain the capsid-encoding regions of FMD virus (FMDV). Ad5 containing serotype A24 capsid sequences (Ad5.A24) has proved to be effective as a vaccine against FMD in livestock species. However, Ad5-vectored FMDV serotype O1 Campos vaccine (Ad5.O1C.2B) provides only partial protection of cattle against homologous challenge. It has been reported that a fiber-modified Ad5 vector expressing Arg-Gly-Asp (RGD) enhances transduction of antigen-presenting cells (APC) in mice. In the current study, we assessed the efficacy of a fiber-modified Ad5 (Adt.O1C.2B.RGD) in cattle. Expression of FMDV capsid proteins was superior in cultured cells infected with the RGD-modified vector. Furthermore, transgene expression of Adt.O1C.2B.RGD was enhanced in cell lines that constitutively express integrin αvβ6, a known receptor for FMDV. In contrast, capsid expression in cattle-derived enriched APC populations was not enhanced by infection with this vector. Our data showed that vaccination with the two vectors yielded similar levels of protection against FMD in cattle. Although none of the vaccinated animals had detectable viremia, FMDV RNA was detected in serum samples from animals with clinical signs. Interestingly, CD4+and CD8+gamma interferon (IFN-γ)+cell responses were detected at significantly higher levels in animals vaccinated with Adt.O1C.2B.RGD than in animals vaccinated with Ad5.O1C.2B. Our results suggest that inclusion of an RGD motif in the fiber of Ad5-vectored FMD vaccine improves transgene delivery and cell-mediated immunity but does not significantly enhance vaccine performance in cattle.

scholarly journals Zinc-binding properties of Junín virus nucleocapsid protein

2001 ◽  
Vol 82 (1) ◽  
pp. 121-128 ◽  
Author(s):  
M. Alejandra Tortorici ◽  
P. Daniel Ghiringhelli ◽  
Mario E. Lozano ◽  
César G. Albariño ◽  
Víctor Romanowski
Keyword(s):  
Nucleocapsid Protein ◽  
Zinc Binding ◽  
Dual Function ◽  
Genome Replication ◽  
Divalent Metal Ions ◽  
Junin Virus ◽  
N Protein ◽  
Carboxy Terminal ◽  
Junín Virus

The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle. First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid. We have expressed the N protein of Junín virus in E. coli and shown that it binds zinc in vitro. This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX2HX23CX4C sequence, which resembles a classical zinc-finger motif. The specificity for zinc binding was demonstrated by competition with other divalent metal ions. The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions. In addition, alternative zinc-binding sites were found.

scholarly journals The Nucleocapsid Protein of Severe Acute Respiratory Syndrome Coronavirus Inhibits Cell Cytokinesis and Proliferation by Interacting with Translation Elongation Factor 1α

2008 ◽  
Vol 82 (14) ◽  
pp. 6962-6971 ◽  
Author(s):  
Bing Zhou ◽  
Junli Liu ◽  
Qiuna Wang ◽  
Xuan Liu ◽  
Xiaorong Li ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Human Peripheral Blood ◽  
Elongation Factor ◽  
Protein Translation ◽  
Peripheral Blood Lymphocytes ◽  
Atypical Pneumonia ◽  
Filamentous Actin ◽  
N Protein

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, an emerging disease characterized by atypical pneumonia. Using a yeast two-hybrid screen with the nucleocapsid (N) protein of SARS-CoV as a bait, the C terminus (amino acids 251 to 422) of the N protein was found to interact with human elongation factor 1-alpha (EF1α), an essential component of the translational machinery with an important role in cytokinesis, promoting the bundling of filamentous actin (F-actin). In vitro and in vivo interaction was then confirmed by immuno-coprecipitation, far-Western blotting, and surface plasmon resonance. It was demonstrated that the N protein of SARS-CoV induces aggregation of EF1α, inhibiting protein translation and cytokinesis by blocking F-actin bundling. Proliferation of human peripheral blood lymphocytes and other human cell lines was significantly inhibited by the infection of recombinant retrovirus expressing SARS-CoV N protein.

scholarly journals RNA Binding Properties of Bunyamwera Virus Nucleocapsid Protein and Selective Binding to an Element in the 5′ Terminus of the Negative-Sense S Segment

2000 ◽  
Vol 74 (21) ◽  
pp. 9946-9952 ◽  
Author(s):  
Jane C. Osborne ◽  
Richard M. Elliott
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Metal Chelate ◽  
Rnase A ◽  
Mobility Shift ◽  
N Protein ◽  
Encapsidation Signal ◽  
Negative Sense ◽  
Bunyamwera Virus

ABSTRACT The genome of Bunyamwera virus (BUN) (familyBunyaviridae, genus Bunyavirus) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5′ terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5′-terminal 32 and 3′-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 μg of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5′ terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.

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