Assembly of Severe Acute Respiratory Syndrome Coronavirus RNA Packaging Signal into Virus-Like Particles Is Nucleocapsid Dependent

Hsieh, Ping-Kun; Chang, Shih‐Chieh; Huang, Chien-Hua; Lee, Ting-Ting; Hsiao, Chiaolong; Kou, Yi-Hen; Chen, I-Yin; Chang, Chung-ke; Huang, Tai-huang; Chang, Shan‐Chwen

doi:10.1128/jvi.79.22.13848-13855.2005

Cited by 193 publications

(242 citation statements)

References 30 publications

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“…Moreover, we confirmed the formation of VLPs in the concentrated culture supernatant using scanning electron microscopy and immunogold-labeling with the anti-S protein polyclonal Ab. The particles were 70 -100 nm in diameter, which is consistent with the sizes as reported previously (18,24,25). The particles were positively stained with immunogold (Fig.…”

Section: Generation Of Recombinant Vv That Expresses the Structural Psupporting

confidence: 77%

“…As shown in Fig. 1E, fraction number 10 contained all the SARS-CoV proteins, and the buoyant density of this fraction was ϳ1.15 g/ml, a value that is consistent with previous reports (18,24,25). Moreover, we confirmed the formation of VLPs in the concentrated culture supernatant using scanning electron microscopy and immunogold-labeling with the anti-S protein polyclonal Ab.…”

Section: Generation Of Recombinant Vv That Expresses the Structural Psupporting

confidence: 77%

“…The supernatants were concentrated ϳ100-fold using the Pellicon XL (cut off molecular weight 3 ϫ 10 5 ; Millipore). The isolation of virus-like particles (VLP) was performed as previously described, with a slight modification (18). Briefly, the concentrated supernatant was placed on 60% (w/w) sucrose cushion and centrifuged at 4.0 ϫ 10 4 rpm for 5 h. The opalescent band was collected and centrifuged in a 20 -60% (w/w) sucrose gradient at 2.7 ϫ 10 4 rpm for 4 h, and then divided into 20 fractions.…”

Section: Confirmation Of Sars-cov-like Particle Formationmentioning

confidence: 99%

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Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV

Yasui¹,

Kai

Kitabatake³

et al. 2008

The Journal of Immunology

251

240

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The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7–8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-β), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.

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Section: Generation Of Recombinant Vv That Expresses the Structural Psupporting

confidence: 77%

Section: Generation Of Recombinant Vv That Expresses the Structural Psupporting

confidence: 77%

Section: Confirmation Of Sars-cov-like Particle Formationmentioning

confidence: 99%

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Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV

Yasui¹,

Kai

Kitabatake³

et al. 2008

The Journal of Immunology

251

240

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“…Huh7 cells (a human hepatoma cell line) and NIH 3T3 cells (a mouse fibroblast cell line) were maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum plus 100 units of penicillin and 100 g of streptomycin/ ml. Expression of recombinant proteins in Huh7 cells was performed by infecting cells with recombinant vaccinia virus (vTF7-3) harboring the T7 RNA polymerase gene, followed by DNA transfection with the Lipofectin reagent as described previously (17). Expression of recombinant proteins in NIH 3T3 cells was performed by DNA transfection with the Lipofectamine 2000 reagent (Invitrogen).…”

Section: Construction Of Expression Plasmids (I)mentioning

confidence: 99%

Differential Requirements of NS4A for Internal NS3 Cleavage and Polyprotein Processing of Hepatitis C Virus

Kou¹,

Chang

Wang³

et al. 2007

J Virol

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The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT 402 ͦS being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.Hepatitis C virus (HCV) is a member of the family Flaviviridae with a single-stranded, positive-sense RNA of 9.6 kb that encodes a polyprotein of approximately 3,000 amino acid residues (8,18,35). The polyprotein precursor is further processed into structural and nonstructural proteins by cellular signal peptidase and viral proteases. Cleavage at the NS2-NS3 junction occurs autoproteolytically with the NS2-3 protease (15), whereas cleavage of the downstream nonstructural proteins is mediated by the viral NS3 serine protease (3,10,14).The viral NS3 protein has multiple functions. The Nterminal-one-third region possesses protease activity, and the C-terminal-two-thirds region contains NTPase and RNA helicase activities. In addition, previous studies indicate that the N-terminal domain of the NS3 protein has the potential for cell transformation (30,41,43). Subcutaneous inoculation of nude mice with cells previously transfected with a plasmid that expresses a C-terminal deletion NS3 protein induced tumor formation (16). Mechanisms of NS3 involved in cell transformation are not fully understood. Nevertheless, the transforming ability of NS3 was abolished when cells were treated with the protease inhibitors phenylmethylsulfonyl fluoride and tosylsulfonyl phenylalanyl chloromethyl ketone (43). This indicates that protease activity is required for the transforming activity of the HCV NS3 protein. Furthermore, internal cleavages of the NS3 protein have been reported....

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“…The formation of the RNP is important for maintaining the RNA in an ordered conformation suitable for replication and transcription of the viral genome [17,[19][20][21]. Previous studies have shown that the CoV N protein is involved in the regulation of cellular processes, such as gene transcription, actin reorganization, host cell cycle progression, and apoptosis [22][23][24][25]. The CoV N protein has also been shown to act as an RNA chaperone [26].…”

Section: Introductionmentioning

confidence: 99%

Oligomerization of the carboxyl terminal domain of the human coronavirus 229E nucleocapsid protein

Lin

Wang³

et al. 2012

FEBS Letters

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102

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a b s t r a c tThe coronavirus (CoV) N protein oligomerizes via its carboxyl terminus. However, the oligomerization mechanism of the C-terminal domains (CTD) of CoV N proteins remains unclear. Based on the protein disorder prediction system, a comprehensive series of HCoV-229E N protein mutants with truncated CTD was generated and systematically investigated by biophysical and biochemical analyses to clarify the role of the C-terminal tail of the HCoV-229E N protein in oligomerization. These results indicate that the last C-terminal tail plays an important role in dimer-dimer association. The C-terminal tail peptide is able to interfere with the oligomerization of the CTD of HCoV-229E N protein and performs the inhibitory effect on viral titre of HCoV-229E. This study may assist the development of anti-viral drugs against HCoV. Structured summary of protein interactions:N and C-terminal tail peptide bind by cosedimentation in solution (View interaction) N and N bind by cosedimentation in solution (View Interaction: 1,2,3,4,5,6,7,8,9,10,11,12) C-terminal tail peptide and N bind by fluorescence technology (View interaction) N and N bind by cross-linking study (View interaction) N and N bind by cross-linking study (View Interaction: 1,2,3,4) Crown

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Assembly of Severe Acute Respiratory Syndrome Coronavirus RNA Packaging Signal into Virus-Like Particles Is Nucleocapsid Dependent

Cited by 193 publications

References 30 publications

Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV

Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV

Differential Requirements of NS4A for Internal NS3 Cleavage and Polyprotein Processing of Hepatitis C Virus

Oligomerization of the carboxyl terminal domain of the human coronavirus 229E nucleocapsid protein

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