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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

Homogeneous sample analysis of recombinant polymerases, RanBP5 and polymerase-RanBP5.Purified product analysis are horizontally grouped for (a) PA-PB1-PB2(1-116), (b) PA-PB1(1-686), (c) RanBP5 and (d) PA-PB1(1-686)-RanBP5. On the left are the Coomassie blue colored SDS PAGE gel of the purified sample with corresponding subunits bands indicated by colored arrows (PA in blue, PB1 in green and RanBP5 in purple). In the middle are the SEC-MALLS chromatograms with the UV signal as a backtrace and molecular weight estimate below the peak. Estimated average molecular weight for each sample is also detailed. SEC-MALLS-RI runs of PA-PB1(1-686), RanBP5 and PA-PB1(1-686)-RanBP5 were performed in the same buffer (50 mM Tris-HCl pH 8.0 and 150 mM NaCl) whereas the SEC-MALLS-RI run of PA-PB1-PB2(1-116) was performed with 50 mM Tris-HCl pH 8.0, 300 mM NaCl and 10% glycerol. (right) corresponding electron microscopy images.
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f2: Homogeneous sample analysis of recombinant polymerases, RanBP5 and polymerase-RanBP5.Purified product analysis are horizontally grouped for (a) PA-PB1-PB2(1-116), (b) PA-PB1(1-686), (c) RanBP5 and (d) PA-PB1(1-686)-RanBP5. On the left are the Coomassie blue colored SDS PAGE gel of the purified sample with corresponding subunits bands indicated by colored arrows (PA in blue, PB1 in green and RanBP5 in purple). In the middle are the SEC-MALLS chromatograms with the UV signal as a backtrace and molecular weight estimate below the peak. Estimated average molecular weight for each sample is also detailed. SEC-MALLS-RI runs of PA-PB1(1-686), RanBP5 and PA-PB1(1-686)-RanBP5 were performed in the same buffer (50 mM Tris-HCl pH 8.0 and 150 mM NaCl) whereas the SEC-MALLS-RI run of PA-PB1-PB2(1-116) was performed with 50 mM Tris-HCl pH 8.0, 300 mM NaCl and 10% glycerol. (right) corresponding electron microscopy images.

Mentions: In order to identify a minimal active core of the human-IAV polymerase, we have systematically dissected the heterotrimeric complex for further characterisation using the polyprotein strategy. The expression of PA-PB1 increases significantly when PB2 is totally removed (compare constructs 1 and 13/15; Table 1). The same observation is made after removing the endonuclease domain (i.e. PA-Nter; compare constructs 14 and 13/15). After purification, PA-PB1 forms homogeneous monomeric and stable particles (Fig. 2b) whereas the complexes with PB2 (constructs 15 and 16) give rise to dimers in solution (Fig. 2a). The dimers are stable enough to withstand salt concentrations up to 2 M NaCl during purification, but the SEC elution peaks are broad suggesting that the oligomerization process is quite dynamic. By adding vRNA promoter-like molecules (e.g. the IAV panhandle)24, we found that the dimers dissociated into monomeric RNA:protein complexes. We were able to show that the truncated PA-PB1 (construct 1) and the PA-PB1-PB2 (construct 14) exhibit expected polymerase activities. The endonuclease activity is similar to that of the isolated PA-Nter domain (supplementary Figure 3), can be inhibited by the same point mutation (i.e. PA-E80A) or compound (i.e. DPBA) and shows comparable dependency on manganese2526. The constructs are also functional in RNA synthesis. Upon addition of a mixture of IAV panhandle, NTPs and [α-32P]-UTP, both truncated PA-PB1 and PA-PB1-PB2 are able to generate an 80-nucleotide long product plus other minor products in absence or in presence of ApG (supplementary Figure 4).


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)

Homogeneous sample analysis of recombinant polymerases, RanBP5 and polymerase-RanBP5.Purified product analysis are horizontally grouped for (a) PA-PB1-PB2(1-116), (b) PA-PB1(1-686), (c) RanBP5 and (d) PA-PB1(1-686)-RanBP5. On the left are the Coomassie blue colored SDS PAGE gel of the purified sample with corresponding subunits bands indicated by colored arrows (PA in blue, PB1 in green and RanBP5 in purple). In the middle are the SEC-MALLS chromatograms with the UV signal as a backtrace and molecular weight estimate below the peak. Estimated average molecular weight for each sample is also detailed. SEC-MALLS-RI runs of PA-PB1(1-686), RanBP5 and PA-PB1(1-686)-RanBP5 were performed in the same buffer (50 mM Tris-HCl pH 8.0 and 150 mM NaCl) whereas the SEC-MALLS-RI run of PA-PB1-PB2(1-116) was performed with 50 mM Tris-HCl pH 8.0, 300 mM NaCl and 10% glycerol. (right) corresponding electron microscopy images.
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Related In: Results  -  Collection

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f2: Homogeneous sample analysis of recombinant polymerases, RanBP5 and polymerase-RanBP5.Purified product analysis are horizontally grouped for (a) PA-PB1-PB2(1-116), (b) PA-PB1(1-686), (c) RanBP5 and (d) PA-PB1(1-686)-RanBP5. On the left are the Coomassie blue colored SDS PAGE gel of the purified sample with corresponding subunits bands indicated by colored arrows (PA in blue, PB1 in green and RanBP5 in purple). In the middle are the SEC-MALLS chromatograms with the UV signal as a backtrace and molecular weight estimate below the peak. Estimated average molecular weight for each sample is also detailed. SEC-MALLS-RI runs of PA-PB1(1-686), RanBP5 and PA-PB1(1-686)-RanBP5 were performed in the same buffer (50 mM Tris-HCl pH 8.0 and 150 mM NaCl) whereas the SEC-MALLS-RI run of PA-PB1-PB2(1-116) was performed with 50 mM Tris-HCl pH 8.0, 300 mM NaCl and 10% glycerol. (right) corresponding electron microscopy images.
Mentions: In order to identify a minimal active core of the human-IAV polymerase, we have systematically dissected the heterotrimeric complex for further characterisation using the polyprotein strategy. The expression of PA-PB1 increases significantly when PB2 is totally removed (compare constructs 1 and 13/15; Table 1). The same observation is made after removing the endonuclease domain (i.e. PA-Nter; compare constructs 14 and 13/15). After purification, PA-PB1 forms homogeneous monomeric and stable particles (Fig. 2b) whereas the complexes with PB2 (constructs 15 and 16) give rise to dimers in solution (Fig. 2a). The dimers are stable enough to withstand salt concentrations up to 2 M NaCl during purification, but the SEC elution peaks are broad suggesting that the oligomerization process is quite dynamic. By adding vRNA promoter-like molecules (e.g. the IAV panhandle)24, we found that the dimers dissociated into monomeric RNA:protein complexes. We were able to show that the truncated PA-PB1 (construct 1) and the PA-PB1-PB2 (construct 14) exhibit expected polymerase activities. The endonuclease activity is similar to that of the isolated PA-Nter domain (supplementary Figure 3), can be inhibited by the same point mutation (i.e. PA-E80A) or compound (i.e. DPBA) and shows comparable dependency on manganese2526. The constructs are also functional in RNA synthesis. Upon addition of a mixture of IAV panhandle, NTPs and [α-32P]-UTP, both truncated PA-PB1 and PA-PB1-PB2 are able to generate an 80-nucleotide long product plus other minor products in absence or in presence of ApG (supplementary Figure 4).

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