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An antigenic epitope of influenza virus nucleoprotein (NP) associated with polymeric forms of NP.

Prokudina EN, Semenova N, Chumakov V, Stitz L - Virol. J. (2008)

Bottom Line: At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope.The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation.Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration.

View Article: PubMed Central - HTML - PubMed

Affiliation: The D.I. Ivanovsky Institute of Virology, Gamaleya str, 16, Moscow, Russia. prokudinaen@mail.ru

ABSTRACT
Intracellular influenza virus nucleoprotein (NP) is characterized by a high efficiency of homo-polymers formation, however their antigenic structure is still incompletely known. Herein, we report that RNase-resistant intracellular NP homo-polymers have a highly ordered conformational antigenic epitope, which depends on inter-subunit interactions of monomeric NPs. Our studies have shown that in radioimmunoprecipitation (RIPA) intracellular NP polymers bind mAb N5D3 and RNase does not prevent their mAb binding. In contrast to NP polymers, NP monomeric subunits, obtained by thermo-dissociation of NP polymers, fail to bind the mAb N5D3 in RIPA. At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope. The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation. Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration. The results obtained suggest that in vivo formation and in vitro renaturation of the N5D3 epitope depend on inter-subunit interactions of monomeric NPs and NP-NP interactions influence the antigenic structure of the influenza virus NP polymers.

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The capacity of polymeric and monomeric NP to bind mAb N5D3. A) RIPA. Radiolabeled cytosol of infected cells was divided into unheated (r.t) portion, containing NP polymers and the heated (70° C) portion, containing NP 56 kDa monomers as a result of NP polymers dissociation. Both unheated and pre-heated portions were subjected to RIPA using polyclonal anti-NP Abs or mAb N5D3. SDS-PAGE of the immunoprecipitates obtained by RIPA using polyclonal Abs (lanes 1, 2) and mAb N5D3 (lanes 3,4). B) Immunolotting. Radiolabeled unheated (1,3) and pre-heated (2,4) cytosols were subjected to SDS-PAGE, followed by Western blot, including electro-transfer onto nitrocellulose membrane, autoradiography and immunodetection using mAb N5D3. Autoradiography (lanes 1, 2) and immunostaining using mAb N5D3 (lanes 3,4) of membrane containing blotted proteins. C) Renaturation of N5D3 epitope caused by self-association of the concentrated soluble NP monomers. The non-concentrated m-NP before RIPA (lane 1) and after immunosorption by RIPA using mAb N5D3 (lane 2). The concentrated soluble self-associated m-NP (as described in the text) before RIPA (lane 3) and after immunosorption by RIPA using mAb N5D3 (lane 4). The aliquot of RIPA immunoprecipitate shown in lane 4 was heated at 100°C for 3 min before SDS-PAGE (lane 5). The samples shown in lanes 1–4 were not additionally pre-heated before SDS-PAGE.
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Figure 1: The capacity of polymeric and monomeric NP to bind mAb N5D3. A) RIPA. Radiolabeled cytosol of infected cells was divided into unheated (r.t) portion, containing NP polymers and the heated (70° C) portion, containing NP 56 kDa monomers as a result of NP polymers dissociation. Both unheated and pre-heated portions were subjected to RIPA using polyclonal anti-NP Abs or mAb N5D3. SDS-PAGE of the immunoprecipitates obtained by RIPA using polyclonal Abs (lanes 1, 2) and mAb N5D3 (lanes 3,4). B) Immunolotting. Radiolabeled unheated (1,3) and pre-heated (2,4) cytosols were subjected to SDS-PAGE, followed by Western blot, including electro-transfer onto nitrocellulose membrane, autoradiography and immunodetection using mAb N5D3. Autoradiography (lanes 1, 2) and immunostaining using mAb N5D3 (lanes 3,4) of membrane containing blotted proteins. C) Renaturation of N5D3 epitope caused by self-association of the concentrated soluble NP monomers. The non-concentrated m-NP before RIPA (lane 1) and after immunosorption by RIPA using mAb N5D3 (lane 2). The concentrated soluble self-associated m-NP (as described in the text) before RIPA (lane 3) and after immunosorption by RIPA using mAb N5D3 (lane 4). The aliquot of RIPA immunoprecipitate shown in lane 4 was heated at 100°C for 3 min before SDS-PAGE (lane 5). The samples shown in lanes 1–4 were not additionally pre-heated before SDS-PAGE.

Mentions: As shown in Fig. 1A the polyclonal antibodies (Abs) reacted in a RIPA with both polymeric NPs, which were present in the unheated cytosol (lane 1), and monomeric 56 kDA NPs, which were a result of thermo-dissociation of NP polymers (lane 2). As also shown NP polymers were recognized by mAb N5D3 in unheated cytosol (lane 3). The pre-treatment of cytosol with RNase did not influence the ability of NP polymers to bind mAb N5D3 (not shown). In contrast to NP polymers, the soluble 56 kDa NP monomers formed after thermo-dissociation of NP polymers were not recognized by mAb N5D3 in a RIPA (lane 4). A trivial explanation could be that the conformational N5D3 epitope is present not only in polymeric NPs but also in monomeric NP subunits, but as a result of the heating process, this epitope is denatured and destroyed. If this assumption is correct, the 56 kDa NP monomers transferred onto nitrocellulose after heating and denaturing SDS-PAGE should not be recognized by mAb N5D3 in a Western blot, as they were not recognized in the heated cytosol by a RIPA (shown in Fig. 1A, lane 4).


An antigenic epitope of influenza virus nucleoprotein (NP) associated with polymeric forms of NP.

Prokudina EN, Semenova N, Chumakov V, Stitz L - Virol. J. (2008)

The capacity of polymeric and monomeric NP to bind mAb N5D3. A) RIPA. Radiolabeled cytosol of infected cells was divided into unheated (r.t) portion, containing NP polymers and the heated (70° C) portion, containing NP 56 kDa monomers as a result of NP polymers dissociation. Both unheated and pre-heated portions were subjected to RIPA using polyclonal anti-NP Abs or mAb N5D3. SDS-PAGE of the immunoprecipitates obtained by RIPA using polyclonal Abs (lanes 1, 2) and mAb N5D3 (lanes 3,4). B) Immunolotting. Radiolabeled unheated (1,3) and pre-heated (2,4) cytosols were subjected to SDS-PAGE, followed by Western blot, including electro-transfer onto nitrocellulose membrane, autoradiography and immunodetection using mAb N5D3. Autoradiography (lanes 1, 2) and immunostaining using mAb N5D3 (lanes 3,4) of membrane containing blotted proteins. C) Renaturation of N5D3 epitope caused by self-association of the concentrated soluble NP monomers. The non-concentrated m-NP before RIPA (lane 1) and after immunosorption by RIPA using mAb N5D3 (lane 2). The concentrated soluble self-associated m-NP (as described in the text) before RIPA (lane 3) and after immunosorption by RIPA using mAb N5D3 (lane 4). The aliquot of RIPA immunoprecipitate shown in lane 4 was heated at 100°C for 3 min before SDS-PAGE (lane 5). The samples shown in lanes 1–4 were not additionally pre-heated before SDS-PAGE.
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Figure 1: The capacity of polymeric and monomeric NP to bind mAb N5D3. A) RIPA. Radiolabeled cytosol of infected cells was divided into unheated (r.t) portion, containing NP polymers and the heated (70° C) portion, containing NP 56 kDa monomers as a result of NP polymers dissociation. Both unheated and pre-heated portions were subjected to RIPA using polyclonal anti-NP Abs or mAb N5D3. SDS-PAGE of the immunoprecipitates obtained by RIPA using polyclonal Abs (lanes 1, 2) and mAb N5D3 (lanes 3,4). B) Immunolotting. Radiolabeled unheated (1,3) and pre-heated (2,4) cytosols were subjected to SDS-PAGE, followed by Western blot, including electro-transfer onto nitrocellulose membrane, autoradiography and immunodetection using mAb N5D3. Autoradiography (lanes 1, 2) and immunostaining using mAb N5D3 (lanes 3,4) of membrane containing blotted proteins. C) Renaturation of N5D3 epitope caused by self-association of the concentrated soluble NP monomers. The non-concentrated m-NP before RIPA (lane 1) and after immunosorption by RIPA using mAb N5D3 (lane 2). The concentrated soluble self-associated m-NP (as described in the text) before RIPA (lane 3) and after immunosorption by RIPA using mAb N5D3 (lane 4). The aliquot of RIPA immunoprecipitate shown in lane 4 was heated at 100°C for 3 min before SDS-PAGE (lane 5). The samples shown in lanes 1–4 were not additionally pre-heated before SDS-PAGE.
Mentions: As shown in Fig. 1A the polyclonal antibodies (Abs) reacted in a RIPA with both polymeric NPs, which were present in the unheated cytosol (lane 1), and monomeric 56 kDA NPs, which were a result of thermo-dissociation of NP polymers (lane 2). As also shown NP polymers were recognized by mAb N5D3 in unheated cytosol (lane 3). The pre-treatment of cytosol with RNase did not influence the ability of NP polymers to bind mAb N5D3 (not shown). In contrast to NP polymers, the soluble 56 kDa NP monomers formed after thermo-dissociation of NP polymers were not recognized by mAb N5D3 in a RIPA (lane 4). A trivial explanation could be that the conformational N5D3 epitope is present not only in polymeric NPs but also in monomeric NP subunits, but as a result of the heating process, this epitope is denatured and destroyed. If this assumption is correct, the 56 kDa NP monomers transferred onto nitrocellulose after heating and denaturing SDS-PAGE should not be recognized by mAb N5D3 in a Western blot, as they were not recognized in the heated cytosol by a RIPA (shown in Fig. 1A, lane 4).

Bottom Line: At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope.The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation.Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration.

View Article: PubMed Central - HTML - PubMed

Affiliation: The D.I. Ivanovsky Institute of Virology, Gamaleya str, 16, Moscow, Russia. prokudinaen@mail.ru

ABSTRACT
Intracellular influenza virus nucleoprotein (NP) is characterized by a high efficiency of homo-polymers formation, however their antigenic structure is still incompletely known. Herein, we report that RNase-resistant intracellular NP homo-polymers have a highly ordered conformational antigenic epitope, which depends on inter-subunit interactions of monomeric NPs. Our studies have shown that in radioimmunoprecipitation (RIPA) intracellular NP polymers bind mAb N5D3 and RNase does not prevent their mAb binding. In contrast to NP polymers, NP monomeric subunits, obtained by thermo-dissociation of NP polymers, fail to bind the mAb N5D3 in RIPA. At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope. The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation. Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration. The results obtained suggest that in vivo formation and in vitro renaturation of the N5D3 epitope depend on inter-subunit interactions of monomeric NPs and NP-NP interactions influence the antigenic structure of the influenza virus NP polymers.

Show MeSH
Related in: MedlinePlus