The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1

doi:10.1016/j.febslet.2005.03.080

Comparative Study

. 2005 May 9;579(12):2623-8.

doi: 10.1016/j.febslet.2005.03.080. Epub 2005 Apr 9.

The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1

Haibin Luo¹, Qing Chen, Jing Chen, Kaixian Chen, Xu Shen, Hualiang Jiang

Affiliations

Affiliation

¹ Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, China.

PMID: 15862300
PMCID: PMC7094256
DOI: 10.1016/j.febslet.2005.03.080

Comparative Study

The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1

Haibin Luo et al. FEBS Lett. 2005.

. 2005 May 9;579(12):2623-8.

doi: 10.1016/j.febslet.2005.03.080. Epub 2005 Apr 9.

Authors

Haibin Luo¹, Qing Chen, Jing Chen, Kaixian Chen, Xu Shen, Hualiang Jiang

Affiliation

¹ Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, China.

PMID: 15862300
PMCID: PMC7094256
DOI: 10.1016/j.febslet.2005.03.080

Abstract

The nucleocapsid (N) protein of SARS coronavirus (SARS_CoV) is a major structural component of virions, which appears to be a multifunctional protein involved in viral RNA replication and translation. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is related to the pre-mRNA splicing in the nucleus and translation regulation in the cytoplasm. In this report, based on the relevant biophysical and biochemical assays, the nucleocapsid protein of SARS_CoV (SARS_N) was discovered to exhibit high binding affinity to human hnRNP A1. GST pull-down results clearly demonstrated that SARS_N protein could directly and specifically bind to human hnRNP A1 in vitro. Yeast two-hybrid assays further indicated in vivo that such binding relates to the fragment (aa 161-210) of SARS_N and the Gly-rich domain (aa 203-320) of hnRNP A1. Moreover, kinetic analyses by surface plasmon resonance (SPR) technology revealed that SARS_N protein has a specific binding affinity against human hnRNP A1 with K(D) at 0.35 +/- 0.02 microM (k(on) = 5.83 +/- 0.42 x 10(3) M(-1)s(-1) and k(off) = 2.06 +/- 0.12 x 10(-3)s(-1)). It is suggested that both SARS_N and hnRNP A1 proteins are possibly within the SARS_CoV replication/transcription complex and SARS_N/human hnRNP A1 interaction might function in the regulation of SARS_CoV RNA synthesis. In addition, the determined results showed that SARS_N protein has only one binding domain for interacting with human hnRNP A1, which is different from the mouse hepatitis virus (MHV) binding case where the nucleocapsid protein of MHV (MHV_N) was found to have two binding domains involved in the MHV_N/hnRNP A1 interaction, thereby suggesting that SARS_N protein might carry out a different binding mode to bind to human hnRNP A1 for its further function performance in comparison with MHV_N.

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Figures

**Figure 1**
SARS_N/human hnRNP A1 interaction determined by GST pull‐down. Samples were analyzed on a 10% SDS–polyacrylamide gel, and the band was visualized with Coomassie brilliant blue. Components in each lane are shown at the top. Lane 1, molecular mass marker; lane 2, purified His‐tagged SARS_N; lane 3, purified GST‐tagged human hnRNP A1; lane 4, agarose gel control; lane 5, human hnRNP A1 and the pull‐down SARS_N.

**Figure 2**
Sensorgrams of human hnRNP A1 binding to the immobilized SARS_N. The binding curves were fitted to 1:1 Langmuir binding model. Superposition of fitting curves (in black) to original curves (in grey) (A) and the small residuals (B) demonstrates the goodness of the fitting.

**Figure 3**
Mapping the interaction domain of SARS_N. Schematic description of the truncated fragments (A) and the yeast two‐hybrid assay results for SARS_N/human hnRNP A1 interactions in their truncated and non‐truncated forms (B). The empty vectors pGBKT7 and pGADT7 co‐transformed were used as the negative control.

**Figure 4**
Interaction of the peptide (ASSRSSSRSRGNSRN) with human hnRNP A1 (A), and the peptide inhibition assay against the human hnRNP A1/SARS_N interaction (B).

**Figure 5**
Comparison of the human hnRNP A1‐binding fragments (the part in frame) in SARS_N with MHV_N.

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References

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[1] Peiris J.S., Chu C.M., Cheng V.C., Chan K.S., Hung I.F., Poon L.L., Law K.I., Tang B.S., Hon T.Y., Chan C.S., Chan K.H., Ng J.S., Zheng B.J., Ng W.L., Lai R.W., Guan Y., Yuen K.Y., Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet, 361, (2003), 1767– 1772. - PMC - PubMed

[2] Peiris J.S., Chu C.M., Cheng V.C., Chan K.S., Hung I.F., Poon L.L., Law K.I., Tang B.S., Hon T.Y., Chan C.S., Chan K.H., Ng J.S., Zheng B.J., Ng W.L., Lai R.W., Guan Y., Yuen K.Y., Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet, 361, (2003), 1767– 1772. - PMC - PubMed

[3] Peiris J.S., Lai S.T., Poon L.L., Guan Y., Yam L.Y., Lim W., Nicholls J., Yee W.K., Yan W.W., Cheung M.T., Cheng V.C., Chan K.H., Tsang D.N., Yung R.W., Ng T.K., Yuen K.Y., Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet, 361, (2003), 1319– 1325. - PMC - PubMed

[4] Peiris J.S., Lai S.T., Poon L.L., Guan Y., Yam L.Y., Lim W., Nicholls J., Yee W.K., Yan W.W., Cheung M.T., Cheng V.C., Chan K.H., Tsang D.N., Yung R.W., Ng T.K., Yuen K.Y., Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet, 361, (2003), 1319– 1325. - PMC - PubMed

[5] Drosten C., Gunther S., Preiser W., Van der Werf S., Brodt H.R., Becker S., Rabenau H., Panning M., Kolesnikova L., Fouchier R.A., Berger A., Burguiere A.M., Cinatl J., Eickmann M., Escriou N., Grywna K., Kramme S., Manuguerra J.C., Muller S., Rickerts V., Sturmer M., Vieth S., Osterhaus A.D., Schmitz H., Doerr H.W., Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med., 348, (2003), 1967– 1976. - PubMed

[6] Drosten C., Gunther S., Preiser W., Van der Werf S., Brodt H.R., Becker S., Rabenau H., Panning M., Kolesnikova L., Fouchier R.A., Berger A., Burguiere A.M., Cinatl J., Eickmann M., Escriou N., Grywna K., Kramme S., Manuguerra J.C., Muller S., Rickerts V., Sturmer M., Vieth S., Osterhaus A.D., Schmitz H., Doerr H.W., Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med., 348, (2003), 1967– 1976. - PubMed

[7] Ksiazek T.G., Erdman D., Goldsmith C.S., Zaki S.R., Peret T., Emery S., Tong S., Urbani C., Comer J.A., Lim W., Rollin P.E., Dowell S.F., Ling A.E., Humphrey C.D., Shieh W.J., Guarner J., Paddock C.D., Rota P., Fields B., DeRisi J., Yang J.Y., Cox N., Hughes J.M., A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med., 348, (2003), 1953– 1966. - PubMed

[8] Ksiazek T.G., Erdman D., Goldsmith C.S., Zaki S.R., Peret T., Emery S., Tong S., Urbani C., Comer J.A., Lim W., Rollin P.E., Dowell S.F., Ling A.E., Humphrey C.D., Shieh W.J., Guarner J., Paddock C.D., Rota P., Fields B., DeRisi J., Yang J.Y., Cox N., Hughes J.M., A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med., 348, (2003), 1953– 1966. - PubMed

[9] Marra M.A., Jones S.J., Astell C.R., Holt R.A., Brooks-Wilson A., Butterfield Y.S., Khattra J., Asano J.K., Barber S.A., Chan S.Y., Cloutier A., Coughlin S.M., Freeman D., Girn N., Griffith O.L., Leach S.R., Mayo M., McDonald H., Montgomery S.B., Pandoh P.K., Petrescu A.S., Robertson A.G., Schein J.E., Siddiqui A., Smailus D.E., Stott J.M., Yang G.S., Plummer F., Andonov A., Artsob H., Bastien N., Bernard K., Booth T.F., Bowness D., Czub M., Drebot M., Fernando L., Flick R., Garbutt M., Gray M., Grolla A., Jones S., Feldmann H., Meyers A., Kabani A., Li Y., Normand S., Stroher U., Tipples G.A., Tyler S., Vogrig R., Ward D., Watson B., Brunham R.C., Krajden M., Petric M., Skowronski D.M., Upton C., Roper R.L., The Genome sequence of the SARS-associated coronavirus. Science, 300, (2003), 1399– 1404. - PubMed

[10] Marra M.A., Jones S.J., Astell C.R., Holt R.A., Brooks-Wilson A., Butterfield Y.S., Khattra J., Asano J.K., Barber S.A., Chan S.Y., Cloutier A., Coughlin S.M., Freeman D., Girn N., Griffith O.L., Leach S.R., Mayo M., McDonald H., Montgomery S.B., Pandoh P.K., Petrescu A.S., Robertson A.G., Schein J.E., Siddiqui A., Smailus D.E., Stott J.M., Yang G.S., Plummer F., Andonov A., Artsob H., Bastien N., Bernard K., Booth T.F., Bowness D., Czub M., Drebot M., Fernando L., Flick R., Garbutt M., Gray M., Grolla A., Jones S., Feldmann H., Meyers A., Kabani A., Li Y., Normand S., Stroher U., Tipples G.A., Tyler S., Vogrig R., Ward D., Watson B., Brunham R.C., Krajden M., Petric M., Skowronski D.M., Upton C., Roper R.L., The Genome sequence of the SARS-associated coronavirus. Science, 300, (2003), 1399– 1404. - PubMed

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The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1

Affiliation

The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1

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