Elucidation of the stability and functional regions of the human coronavirus OC43 nucleocapsid protein

Huang, Chun Fa; Hsu, Yen Lan; Chiang, Wan Ling; Hou, Ming-Hon

doi:10.1002/pro.225

Cited by 26 publications

(26 citation statements)

References 51 publications

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“…Previous studies showed that the coronaviral N protein has a high affinity for the TRS sequence (Grossoehme et al, 2009). A repeated TRS sequence has been used as a probe in the SPR experiments to measure the interaction of the N protein with viral RNA (Huang et al, 2009;Nelson et al, 2000). The applied viral RNA probe consisted of repeated IBV TRS sequences, 5 0 -(CUUAA-CAA) 4 -3 0 and was biotin-labeled.…”

Section: Functional Test Of the Positively Selected Sitesmentioning

confidence: 99%

Evolution of infectious bronchitis virus in Taiwan: Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity

Kuo

Kao

Hou

et al. 2013

Veterinary Microbiology

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RNA recombination has been shown to underlie the sporadic emergence of new variants of coronavirus, including the infectious bronchitis virus (IBV), a highly contagious avian pathogen. We have demonstrated that RNA recombination can give rise to a new viral population, supported by the finding that most isolated Taiwanese (TW) IBVs, similar to Chinese (CH) IBVs, exhibit a genetic rearrangement with the American (US) IBV at the 5' end of the nucleocapsid (N) gene. Here, we further show that positive selection has occurred at two sites within the putative crossover region of the N-terminal domain (NTD) of the TW IBV N protein. Based on the crystal structure of the NTD, the stereographic positions of both predicted selected sites do not fall close to the RNA-binding groove. Surprisingly, converting either of the two residues to the amino acid present in most CH IBVs resulted in significantly reduced affinity of the N protein for the synthetic RNA repeats of the viral transcriptional regulatory sequence. These results suggest that modulating the amino acid residue at either selected site may alter the conformation of the N protein and affect the viral RNA-N interaction. This study illustrates that the N protein of the current TW IBV variant has been shaped by both RNA recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the RNA-binding capacity of the N protein.

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Section: Functional Test Of the Positively Selected Sitesmentioning

confidence: 99%

Evolution of infectious bronchitis virus in Taiwan: Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity

Kuo

Kao

Hou

et al. 2013

Veterinary Microbiology

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“…Self-association of the N protein is an important step in virus particle assembly for many CoVs [32]. Previous studies have shown that full-length CoV N proteins can form high-order oligomers, and the C-terminal domains of the CoV N proteins are responsible for oligomerization [30,[33][34][35][36][37][38]. Crystal structures of the C-terminal domains of CoV N proteins have been published and suggest that the basic building block for coronavirus nucleocapsid formation is the dimeric assembly of the N protein [34,[39][40][41].…”

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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“…In the Type II immunoreactivity pattern, the immunodominant region was located in the aa 174-300 region, while in the Type III immunoreactivity pattern, all of the three structural regions showed strong antigenicity. According to previous circular dichroism and SPR studies, the central region is necessary for the disorder conformation and has been shown to have RNA-binding activity (Huang et al, 2009).…”

Section: Discussionmentioning

confidence: 96%

“…The yield from 1 l of bacterial culture was as much as 10 mg of pure NP after extraction and column chromatography. The ability of the recombinant HCoV-OC43 protein to bind the intergenic sequence of the RNA genomic leader by SPR was examined (Huang et al, 2009). These results, which showed efficient binding of HCoV-OC43 to the RNA, suggest that the purified recombinant NP was folded properly.…”

Section: Discussionmentioning

confidence: 99%

“…The NP is responsible for recognising a stretch of RNA that serves as a viral packaging signal, leading to the formation of the ribonucleoprotein (RNP) complex during viral assembly (Lai and Cavanagh, 1997). Previous surface plasmon resonance (SPR) data showed that purified recombinant HCoV-OC43 NP can bind single-stranded RNA, suggesting that recombinant HCoV-OC43 NP is folded properly (Huang et al, 2009).…”

Section: Purification Of Recombinant Hcov-oc43 Npmentioning

confidence: 99%

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Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: Implications for the diagnosis of coronavirus infection

Fang-Ying

Lin

Ying

et al. 2013

Journal of Virological Methods

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Previous studies have reported that a prokaryotic-expressed recombinant nucleocapsid protein (NP) is a suitable reagent for the epidemiological screening of coronavirus infection. In this study, soluble recombinant human coronavirus OC43 (HCoV-OC43) NP was produced to examine the antigenicity of the HCoV-OC43 NP of betacoronavirus. Using the purified recombinant NP as an antigen, a polyclonal antibody from rabbit serum with specificity for HCoV-OC43 NP was generated; this antibody reacts specifically with HCoV-OC43 NP and does not cross-react with other human CoV NPs (including those of SARS-CoV and HCoV-229E) by Western blot. Sera from 26 young adults, 17 middle-aged and elderly patients with respiratory infection, and 15 cord blood samples were also tested. Strong reactivity to the NPs of HCoV-OC43 was observed in 96%, 82%, and 93% of the serum samples from the young adults, respiratory patients, and cord blood samples, respectively. To identify the immunoreactivities of the three structural regions of the NP that are recognised by the rabbit polyclonal antibody and human serum, the antigenicities of three protein fragments, including the N-terminal domain (aa 1-173), the central-linker region (aa 174-300), and the C-terminal domain (aa 301-448), were evaluated by Western blot. The rabbit polyclonal antibody demonstrated greater immunoreactivity to the central-linker region and the C-terminal domain than to the N-terminal domain. Three different patterns for the immunoreactivities of the three structural regions of HCoV-OC43 NP were observed in human serum, suggesting variability in the immune responses that occur during HCoV-OC43 infection in humans. The central-linker region of the NP appeared to be the most highly immunoreactive region for all three patterns observed. The goal of this study was to offer insight into the design of diagnostic tools for HCoV infection.

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Elucidation of the stability and functional regions of the human coronavirus OC43 nucleocapsid protein

Cited by 26 publications

References 51 publications

Evolution of infectious bronchitis virus in Taiwan: Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity

Evolution of infectious bronchitis virus in Taiwan: Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity

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

Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: Implications for the diagnosis of coronavirus infection

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