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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

vRNA binding and specificity.(a) Binding titration of the truncated trimer PA-PB1-PB2(1-116) towards the 5′-vRNAp (blue triangle) and 3′-vRNAp (red square) sequences using fluorescence anisotropy at 300 mM NaCl. (b) Binding titration performed by filter binding assay against P32 labelled 5′-vRNAp (blue triangle) and 3′-vRNAp (red square) using 300 mM NaCl. Bound RNA fraction is plotted as a function of polymerase concentration. (c) Binding titration of the truncated dimer PA-PB1(1-686) performed at 150 and 300 mM NaCl against the 5-′vRNAp (dark and light blue triangles), 3′-vRNAp (orange and dark red squares) and polyUC RNA (light and dark green circles) by fluorescence anisotropy. (d) Binding titration of different polymerases and polymerase-RanBP5 constructs against the 5′-vRNAp at 300 mM NaCl by fluorescence anisotropy. PA-PB1-PB2(116) and PA-PB1(1-686) are depicted by blue and orange triangles respectively, PA-PB1(1-686)-RanBP5 is depicted with purple triangles. For all anisotropy titrations (a,c,d) subtracted anisotropy is plotted as a function of protein concentration.
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f3: vRNA binding and specificity.(a) Binding titration of the truncated trimer PA-PB1-PB2(1-116) towards the 5′-vRNAp (blue triangle) and 3′-vRNAp (red square) sequences using fluorescence anisotropy at 300 mM NaCl. (b) Binding titration performed by filter binding assay against P32 labelled 5′-vRNAp (blue triangle) and 3′-vRNAp (red square) using 300 mM NaCl. Bound RNA fraction is plotted as a function of polymerase concentration. (c) Binding titration of the truncated dimer PA-PB1(1-686) performed at 150 and 300 mM NaCl against the 5-′vRNAp (dark and light blue triangles), 3′-vRNAp (orange and dark red squares) and polyUC RNA (light and dark green circles) by fluorescence anisotropy. (d) Binding titration of different polymerases and polymerase-RanBP5 constructs against the 5′-vRNAp at 300 mM NaCl by fluorescence anisotropy. PA-PB1-PB2(116) and PA-PB1(1-686) are depicted by blue and orange triangles respectively, PA-PB1(1-686)-RanBP5 is depicted with purple triangles. For all anisotropy titrations (a,c,d) subtracted anisotropy is plotted as a function of protein concentration.

Mentions: We used fluorescence anisotropy to measure the interaction between our truncated polymerase constructs and the conserved vRNA ends by following the fluorescence polarization increase of a fluorescently labelled RNA when it binds the polymerase. For this purpose, we used 5′-vRNAp and 3′-vRNAp, both labelled with fluorescein amidite (FAM) at the opposite extremity of the putative interaction, i.e. at the 3′-end for the 5′-vRNAp and vice versa (Fig. 3 and Table 2). At 300 mM NaCl, the Kd for the 5′-vRNAp are similar (sub-nanomolar) for both constructs 1 and 14, whereas for the 3′-vRNAp, a Kd of 36 nM for the construct with PB2 is obtained (Fig. 3a) and no binding was observed for the construct without PB2 (Fig. 3c). As the estimated Kd for the 5′-end was in a range below the working concentration and beyond the sensitivity limit of the fluorescence anisotropy measurement, another method was required to determine the 5′-vRNAp affinity. Filter binding assays (FBA) were then therefore used for precise Kd determination (Fig. 3b). With the corresponding radioactive probes, we found values of 0.2–0.4 nM for the 5′-end. For the 3′-end, the Kd was 100-fold higher and critically dependent on the presence of PB2. Salt concentration is also an important parameter. We show different binding characteristics at 150 and 300 mM NaCl of the PA-PB1 towards different RNAs. The Kd value of PA-PB1 for the 5′-end remains sub-nanomolar in the range of 150 to 300 mM NaCl. In contrast, at 150 mM NaCl, 3′-vRNAp binds to PA-PB1 with the same affinity than for a poly-UC (Fig. 3c), and so is non-specific. All relevant titrations were performed at 300 mM NaCl to prevent non-specific affinity measurements. Recently published affinity data33 working at 500 mM NaCl likewise consistent with very high affinity of the 5′-vRNAp towards IAV polymerase but this study failed to measure 3′-vRNAp binding within the nanomolar range, indicating that 3′-vRNAp binding is also tightly influenced by salt concentration.


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)

vRNA binding and specificity.(a) Binding titration of the truncated trimer PA-PB1-PB2(1-116) towards the 5′-vRNAp (blue triangle) and 3′-vRNAp (red square) sequences using fluorescence anisotropy at 300 mM NaCl. (b) Binding titration performed by filter binding assay against P32 labelled 5′-vRNAp (blue triangle) and 3′-vRNAp (red square) using 300 mM NaCl. Bound RNA fraction is plotted as a function of polymerase concentration. (c) Binding titration of the truncated dimer PA-PB1(1-686) performed at 150 and 300 mM NaCl against the 5-′vRNAp (dark and light blue triangles), 3′-vRNAp (orange and dark red squares) and polyUC RNA (light and dark green circles) by fluorescence anisotropy. (d) Binding titration of different polymerases and polymerase-RanBP5 constructs against the 5′-vRNAp at 300 mM NaCl by fluorescence anisotropy. PA-PB1-PB2(116) and PA-PB1(1-686) are depicted by blue and orange triangles respectively, PA-PB1(1-686)-RanBP5 is depicted with purple triangles. For all anisotropy titrations (a,c,d) subtracted anisotropy is plotted as a function of protein concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: vRNA binding and specificity.(a) Binding titration of the truncated trimer PA-PB1-PB2(1-116) towards the 5′-vRNAp (blue triangle) and 3′-vRNAp (red square) sequences using fluorescence anisotropy at 300 mM NaCl. (b) Binding titration performed by filter binding assay against P32 labelled 5′-vRNAp (blue triangle) and 3′-vRNAp (red square) using 300 mM NaCl. Bound RNA fraction is plotted as a function of polymerase concentration. (c) Binding titration of the truncated dimer PA-PB1(1-686) performed at 150 and 300 mM NaCl against the 5-′vRNAp (dark and light blue triangles), 3′-vRNAp (orange and dark red squares) and polyUC RNA (light and dark green circles) by fluorescence anisotropy. (d) Binding titration of different polymerases and polymerase-RanBP5 constructs against the 5′-vRNAp at 300 mM NaCl by fluorescence anisotropy. PA-PB1-PB2(116) and PA-PB1(1-686) are depicted by blue and orange triangles respectively, PA-PB1(1-686)-RanBP5 is depicted with purple triangles. For all anisotropy titrations (a,c,d) subtracted anisotropy is plotted as a function of protein concentration.
Mentions: We used fluorescence anisotropy to measure the interaction between our truncated polymerase constructs and the conserved vRNA ends by following the fluorescence polarization increase of a fluorescently labelled RNA when it binds the polymerase. For this purpose, we used 5′-vRNAp and 3′-vRNAp, both labelled with fluorescein amidite (FAM) at the opposite extremity of the putative interaction, i.e. at the 3′-end for the 5′-vRNAp and vice versa (Fig. 3 and Table 2). At 300 mM NaCl, the Kd for the 5′-vRNAp are similar (sub-nanomolar) for both constructs 1 and 14, whereas for the 3′-vRNAp, a Kd of 36 nM for the construct with PB2 is obtained (Fig. 3a) and no binding was observed for the construct without PB2 (Fig. 3c). As the estimated Kd for the 5′-end was in a range below the working concentration and beyond the sensitivity limit of the fluorescence anisotropy measurement, another method was required to determine the 5′-vRNAp affinity. Filter binding assays (FBA) were then therefore used for precise Kd determination (Fig. 3b). With the corresponding radioactive probes, we found values of 0.2–0.4 nM for the 5′-end. For the 3′-end, the Kd was 100-fold higher and critically dependent on the presence of PB2. Salt concentration is also an important parameter. We show different binding characteristics at 150 and 300 mM NaCl of the PA-PB1 towards different RNAs. The Kd value of PA-PB1 for the 5′-end remains sub-nanomolar in the range of 150 to 300 mM NaCl. In contrast, at 150 mM NaCl, 3′-vRNAp binds to PA-PB1 with the same affinity than for a poly-UC (Fig. 3c), and so is non-specific. All relevant titrations were performed at 300 mM NaCl to prevent non-specific affinity measurements. Recently published affinity data33 working at 500 mM NaCl likewise consistent with very high affinity of the 5′-vRNAp towards IAV polymerase but this study failed to measure 3′-vRNAp binding within the nanomolar range, indicating that 3′-vRNAp binding is also tightly influenced by salt concentration.

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