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Structural characterization of recombinant IAV polymerase reveals a stable complex between viral PA-PB1 heterodimer and host RanBP5.

Swale C, Monod A, Tengo L, Labaronne A, Garzoni F, Bourhis JM, Cusack S, Schoehn G, Berger I, Ruigrok RW, Crépin T - Sci Rep (2016)

Bottom Line: In contrast, 3'-vRNA recognition critically depends on the PB2 N-terminal domain.Moreover, we demonstrate that PA-PB1 forms a stable and stoichiometric complex with host nuclear import factor RanBP5 that can be modelled using SAXS and we show that the PA-PB1-RanPB5 complex is no longer capable of 5'-vRNA binding.Our results provide further evidence for a step-wise assembly of IAV structural components, regulated by nuclear transport mechanisms and host factor binding.

View Article: PubMed Central - PubMed

Affiliation: Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.

ABSTRACT
The genome of influenza A virus (IAV) comprises eight RNA segments (vRNA) which are transcribed and replicated by the heterotrimeric IAV RNA-dependent RNA-polymerase (RdRp). RdRp consists of three subunits (PA, PB1 and PB2) and binds both the highly conserved 3'- and 5'-ends of the vRNA segment. The IAV RdRp is an important antiviral target, but its structural mechanism has remained largely elusive to date. By applying a polyprotein strategy, we produced RdRp complexes and define a minimal human IAV RdRp core complex. We show that PA-PB1 forms a stable heterodimeric submodule that can strongly interact with 5'-vRNA. In contrast, 3'-vRNA recognition critically depends on the PB2 N-terminal domain. Moreover, we demonstrate that PA-PB1 forms a stable and stoichiometric complex with host nuclear import factor RanBP5 that can be modelled using SAXS and we show that the PA-PB1-RanPB5 complex is no longer capable of 5'-vRNA binding. Our results provide further evidence for a step-wise assembly of IAV structural components, regulated by nuclear transport mechanisms and host factor binding.

No MeSH data available.


Related in: MedlinePlus

Online SAXS analysis of PA-PB1, RanBP5 and PA-PB1-RanBP5 complex in solution.Results are shown for (a) PA-PB1(1-686), (b) RanBP5 and (c) PA-PB1-RanBP5. On the left are the experimental Ln[I(q)] curves with the ab-initio DAMMIN curve fit (blue) using the Damstart (DAMAVER) as an initial constraint and the CRYSOL fit (red) of the closest homologous X-ray structure. The MONSA fit is also shown for PA-PB1-RanBP5 in black. The Chi2, to evaluate the statistical similarity between experimental intensities and those computed from a model, are also shown. On the right, the homologous PDB structure of PA-PB1(1-686) from Bat-IAV polymerase is depicted in cartoon and fitted in both the DAMAVER and the MONSA generated envelops of PA-PB1(1-686) which are depicted in grey. PA is coloured in blue, PB1 in green and the NLS of PB1 in magenta. The DAMAVER and MONSA envelop of RanBp5 is coloured in purple. As a comparison, the atomic structure of Kap123P (pdb is: 3W3T) is depicted in cartoon with a sky blue colour alongside the DAMAVER envelop of RanBP5. All envelopes and models are adjusted at the same size scale.
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f4: Online SAXS analysis of PA-PB1, RanBP5 and PA-PB1-RanBP5 complex in solution.Results are shown for (a) PA-PB1(1-686), (b) RanBP5 and (c) PA-PB1-RanBP5. On the left are the experimental Ln[I(q)] curves with the ab-initio DAMMIN curve fit (blue) using the Damstart (DAMAVER) as an initial constraint and the CRYSOL fit (red) of the closest homologous X-ray structure. The MONSA fit is also shown for PA-PB1-RanBP5 in black. The Chi2, to evaluate the statistical similarity between experimental intensities and those computed from a model, are also shown. On the right, the homologous PDB structure of PA-PB1(1-686) from Bat-IAV polymerase is depicted in cartoon and fitted in both the DAMAVER and the MONSA generated envelops of PA-PB1(1-686) which are depicted in grey. PA is coloured in blue, PB1 in green and the NLS of PB1 in magenta. The DAMAVER and MONSA envelop of RanBp5 is coloured in purple. As a comparison, the atomic structure of Kap123P (pdb is: 3W3T) is depicted in cartoon with a sky blue colour alongside the DAMAVER envelop of RanBP5. All envelopes and models are adjusted at the same size scale.

Mentions: Online SEC-SAXS was used for the characterisation of PA-PB1(1-686), RanBP5 and PA-PB1(1-686)-RanBP5, respectively in solution. Using the Vc determination method39 on the diffusion data of PA-PB1(1-686) shows a Mw estimate of 146 kDa (Table 3 and supplementary Figure 7). The calculated Mw is 166 kDa. The Guinier transform measures a hydrodynamic radius (Rg) of 36.2 Å and GNOM produces the pair distribution function fit with a Dmax of 128 Å. Further analysis of the SAXS curve (Fig. 4a) shows a visually adequate correlation with the CRYSOL curve which uses the bat-IAV polymerase structure (PDB id: 4WSB) as a model. To evaluate the statistical similarity between experimental intensities and those computed from a model has been derived. The high chi2 values are observed for this dataset, due to a noisy SAXS curve. A slight deviation of the fit is observed above a q range of 0.8 nm−1, suggesting conformational differences between the scattering curve and the crystal structure coordinates. Ab-initio modelling was performed using 15 DAMMIF models which were averaged by DAMAVER. Averaged model correlation with the diffusion curve was then checked using the damstart file as a starting envelope for DAMMIN. The polymerase DAMAVER envelope (Fig. 4a) appears as a pear shaped structure in which the homologous model can be fitted. Additional envelope volume is visible close to the endonuclease domain, implying that it adopts multiple conformations in solution.


Structural characterization of recombinant IAV polymerase reveals a stable complex between viral PA-PB1 heterodimer and host RanBP5.

Swale C, Monod A, Tengo L, Labaronne A, Garzoni F, Bourhis JM, Cusack S, Schoehn G, Berger I, Ruigrok RW, Crépin T - Sci Rep (2016)

Online SAXS analysis of PA-PB1, RanBP5 and PA-PB1-RanBP5 complex in solution.Results are shown for (a) PA-PB1(1-686), (b) RanBP5 and (c) PA-PB1-RanBP5. On the left are the experimental Ln[I(q)] curves with the ab-initio DAMMIN curve fit (blue) using the Damstart (DAMAVER) as an initial constraint and the CRYSOL fit (red) of the closest homologous X-ray structure. The MONSA fit is also shown for PA-PB1-RanBP5 in black. The Chi2, to evaluate the statistical similarity between experimental intensities and those computed from a model, are also shown. On the right, the homologous PDB structure of PA-PB1(1-686) from Bat-IAV polymerase is depicted in cartoon and fitted in both the DAMAVER and the MONSA generated envelops of PA-PB1(1-686) which are depicted in grey. PA is coloured in blue, PB1 in green and the NLS of PB1 in magenta. The DAMAVER and MONSA envelop of RanBp5 is coloured in purple. As a comparison, the atomic structure of Kap123P (pdb is: 3W3T) is depicted in cartoon with a sky blue colour alongside the DAMAVER envelop of RanBP5. All envelopes and models are adjusted at the same size scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837377&req=5

f4: Online SAXS analysis of PA-PB1, RanBP5 and PA-PB1-RanBP5 complex in solution.Results are shown for (a) PA-PB1(1-686), (b) RanBP5 and (c) PA-PB1-RanBP5. On the left are the experimental Ln[I(q)] curves with the ab-initio DAMMIN curve fit (blue) using the Damstart (DAMAVER) as an initial constraint and the CRYSOL fit (red) of the closest homologous X-ray structure. The MONSA fit is also shown for PA-PB1-RanBP5 in black. The Chi2, to evaluate the statistical similarity between experimental intensities and those computed from a model, are also shown. On the right, the homologous PDB structure of PA-PB1(1-686) from Bat-IAV polymerase is depicted in cartoon and fitted in both the DAMAVER and the MONSA generated envelops of PA-PB1(1-686) which are depicted in grey. PA is coloured in blue, PB1 in green and the NLS of PB1 in magenta. The DAMAVER and MONSA envelop of RanBp5 is coloured in purple. As a comparison, the atomic structure of Kap123P (pdb is: 3W3T) is depicted in cartoon with a sky blue colour alongside the DAMAVER envelop of RanBP5. All envelopes and models are adjusted at the same size scale.
Mentions: Online SEC-SAXS was used for the characterisation of PA-PB1(1-686), RanBP5 and PA-PB1(1-686)-RanBP5, respectively in solution. Using the Vc determination method39 on the diffusion data of PA-PB1(1-686) shows a Mw estimate of 146 kDa (Table 3 and supplementary Figure 7). The calculated Mw is 166 kDa. The Guinier transform measures a hydrodynamic radius (Rg) of 36.2 Å and GNOM produces the pair distribution function fit with a Dmax of 128 Å. Further analysis of the SAXS curve (Fig. 4a) shows a visually adequate correlation with the CRYSOL curve which uses the bat-IAV polymerase structure (PDB id: 4WSB) as a model. To evaluate the statistical similarity between experimental intensities and those computed from a model has been derived. The high chi2 values are observed for this dataset, due to a noisy SAXS curve. A slight deviation of the fit is observed above a q range of 0.8 nm−1, suggesting conformational differences between the scattering curve and the crystal structure coordinates. Ab-initio modelling was performed using 15 DAMMIF models which were averaged by DAMAVER. Averaged model correlation with the diffusion curve was then checked using the damstart file as a starting envelope for DAMMIN. The polymerase DAMAVER envelope (Fig. 4a) appears as a pear shaped structure in which the homologous model can be fitted. Additional envelope volume is visible close to the endonuclease domain, implying that it adopts multiple conformations in solution.

Bottom Line: In contrast, 3'-vRNA recognition critically depends on the PB2 N-terminal domain.Moreover, we demonstrate that PA-PB1 forms a stable and stoichiometric complex with host nuclear import factor RanBP5 that can be modelled using SAXS and we show that the PA-PB1-RanPB5 complex is no longer capable of 5'-vRNA binding.Our results provide further evidence for a step-wise assembly of IAV structural components, regulated by nuclear transport mechanisms and host factor binding.

View Article: PubMed Central - PubMed

Affiliation: Université Grenoble Alpes, Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 71 avenue des Martyrs, CS 90181, F-38042 Grenoble Cedex 9, France.

ABSTRACT
The genome of influenza A virus (IAV) comprises eight RNA segments (vRNA) which are transcribed and replicated by the heterotrimeric IAV RNA-dependent RNA-polymerase (RdRp). RdRp consists of three subunits (PA, PB1 and PB2) and binds both the highly conserved 3'- and 5'-ends of the vRNA segment. The IAV RdRp is an important antiviral target, but its structural mechanism has remained largely elusive to date. By applying a polyprotein strategy, we produced RdRp complexes and define a minimal human IAV RdRp core complex. We show that PA-PB1 forms a stable heterodimeric submodule that can strongly interact with 5'-vRNA. In contrast, 3'-vRNA recognition critically depends on the PB2 N-terminal domain. Moreover, we demonstrate that PA-PB1 forms a stable and stoichiometric complex with host nuclear import factor RanBP5 that can be modelled using SAXS and we show that the PA-PB1-RanPB5 complex is no longer capable of 5'-vRNA binding. Our results provide further evidence for a step-wise assembly of IAV structural components, regulated by nuclear transport mechanisms and host factor binding.

No MeSH data available.


Related in: MedlinePlus