The Nucleocapsid Protein of Human Coronavirus NL63

Zuwała, Kaja; Gołda, Anna; Kabala, Wojciech; Burmistrz, Michał; Zdzalik, Michal; Nowak, Paulina; Kędracka–Krok, Sylwia; Zarębski, Mirosław; Dobrucki, Jurek; Florek, Dominik; Żegleń, Sławomir; Wojarski, Jacek; Potempa, Jan; Dubin, Grzegorz; Pyrć, Krzysztof

doi:10.1371/journal.pone.0117833

Cited by 22 publications

(29 citation statements)

References 77 publications

(83 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both domains were obtained with high efficiency (ϳ30 mg/liter of culture) and purified to homogeneity. Size exclusion chromatography revealed that the NTD was present in a monomeric form in solution, whereas the CTD had eluted as a dimer (and also as highmolecular-weight aggregates that were unstable at higher concentrations), consistent with previously published data (32). The NTD crystallized readily, but we failed to obtain CTD crystals from the initial construct.…”

Section: Resultssupporting

confidence: 88%

Structural Characterization of Human Coronavirus NL63 N Protein

Szelążek

Kabala

Kuś

et al. 2017

J Virol

Self Cite

View full text Add to dashboard Cite

Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N-and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections.

show abstract

Section: Resultssupporting

confidence: 88%

Structural Characterization of Human Coronavirus NL63 N Protein

Szelążek

Kabala

Kuś

et al. 2017

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…We suggest that APOBEC3 proteins may affect coronavirus replication by two mechanisms: first, by deamination of yet unidentified cellular targets or deamination of single sites in the coronaviral genome; and second, by direct, non-catalytic interaction with viral RNA and/or proteins. APOBEC proteins may interact with the coronaviral N protein, which is an essential structural protein for formation of the viral ribonucleocapsid, and which also has a role in virus replication [38][39][40][41][42] . Our results demonstrated that all APOBEC3 proteins with inhibitory activity bound to the N protein.…”

Section: Discussionmentioning

confidence: 99%

“…Samples were centrifuged (10 min at 12,000 × g), pelleted cell debris was discarded and resulting supernatants were stored at −80 °C. HCoV-NL63 N protein was prepared as described previously 39 . Cell lysates were incubated Sense ACG CGA ATT CCA CCA TGG AAG CCA GCC CAG CAT CCG G Antisense GAC TGC GGC CGC CTA TCA AGC GTA ATC TGG AAC ATC GTA TGG GTA GTT TCC CTG ATT CTG GAG AA A3C Sense ACG CGA ATT CCA CCA TGA ATC CAC AGA TCA GAA ACC CGA TG Antisense GAC TGC GGC CGC CTA TCA AGC GTA ATC TGG AAC ATC GTA TGG GTA CTG GAG ACT CTC CCG TAG for 1 h at 37 °C with the purified N protein in 1 × activity buffer, containing 100 mM KCl, 10 mM HEPES (pH 7.4), 100 µM ZnCl 2 , 1 mM DTT and 1 mM EDTA 17 .…”

Section: Hcov-nl63mentioning

confidence: 99%

“…APOBEC3 proteins interact with human coronavirus (HCoV)-NL63 nucleoprotein (N). (a) 293T cell lysates enriched in active APOBEC3 proteins (A3C, A3F or A3H) or their catalytically inactive derivatives (mA3C, mA3F or mA3H) were incubated with purified HCoV-NL63 N (produced in Escherichia coli)39 for 1 h at 37 °C. Subsequently, agarose spheres coated with anti-HA antibodies were added, and proteins were co-precipitated using spin columns.…”

mentioning

confidence: 99%

See 1 more Smart Citation

APOBEC3-mediated restriction of RNA virus replication

Milewska

Kindler

V’kovski

et al. 2018

Sci Rep

Self Cite

103

100

View full text Add to dashboard Cite

APOBEC3 family members are cytidine deaminases with roles in intrinsic responses to infection by retroviruses and retrotransposons, and in the control of other DNA viruses, such as herpesviruses, parvoviruses and hepatitis B virus. Although effects of APOBEC3 members on viral DNA have been demonstrated, it is not known whether they edit RNA genomes through cytidine deamination. Here, we investigated APOBEC3-mediated restriction of Coronaviridae. In experiments in vitro, three human APOBEC3 proteins (A3C, A3F and A3H) inhibited HCoV-NL63 infection and limited production of progeny virus, but did not cause hypermutation of the coronaviral genome. APOBEC3-mediated restriction was partially dependent on enzyme activity, and was reduced by the use of enzymatically inactive APOBEC3. Moreover, APOBEC3 proteins bound to the coronaviral nucleoprotein, and this interaction also affected viral replication. Although the precise molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive, our results further our understanding of APOBEC-mediated restriction of RNA virus infections.

show abstract

“…In agreement with our previous studies, the M protein migrated as two bands, reflecting its two different glycosylation states. Similarly, two bands of the N protein were observed, upon electrophoresis, resulting from protein degradation under denaturing conditions (23). In contrast, the apparent molecular weight of the S protein band was higher than expected, most likely because of the glycosylation and/or incomplete denaturation of the protein trimer.…”

Section: Resultsmentioning

confidence: 79%

Membrane Protein of Human Coronavirus NL63 Is Responsible for Interaction with the Adhesion Receptor

Naskalska

Dąbrowska

Szczepański

et al. 2019

J Virol

Self Cite

View full text Add to dashboard Cite

Human coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of HCoV-NL63 mediates this attachment. Using viruslike particles lacking the spike (S) protein, we demonstrate that binding to the cell is not S protein dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly, in silico analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation. IMPORTANCE It is generally accepted that the coronaviral S protein is responsible for viral interaction with a cellular receptor. Here we show that the M protein is also an important player during early stages of HCoV-NL63 infection and that the concerted action of the two proteins (M and S) is a prerequisite for effective infection. We believe that this study broadens the understanding of HCoV-NL63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. The data presented here may also be important for future research into vaccine or drug development.

show abstract

The Nucleocapsid Protein of Human Coronavirus NL63

Cited by 22 publications

References 77 publications

Structural Characterization of Human Coronavirus NL63 N Protein

Structural Characterization of Human Coronavirus NL63 N Protein

APOBEC3-mediated restriction of RNA virus replication

Membrane Protein of Human Coronavirus NL63 Is Responsible for Interaction with the Adhesion Receptor

Contact Info

Product

Resources

About