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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

Truncated human-IAV RdRp polyprotein expression.(a) Logic of the TEV-PA-PB1-PB2-CFP polyprotein expression in a schematic view. During expression, TEV will process the polyprotein ensuring a stoichiometric assembly of PA, PB1 and PB2. YFP and CFP are produced during the process and monitor respectively baculovirus proliferation and polyprotein translation. (b) Truncated RdRp constructs where PB2 is incrementally extended until residues 116, 320 or 483. (c) YFP (left) and CFP (right) fluorescence kinetics measured during Hi-5 insect cells culture of truncated RdRp constructs. YFP (λexi = 488 nm, λemi = 525 nm) and CFP (λexi = 430 nm, λemi = 480 nm) measurements were performed on cellular extracts prepared by sonicating 1 × 106 cells in PBS (500 μL) follow by centrifugation. Fluorescence intensities are plotted against time after infection. (d) Small scale nickel resin purification analysis by western blot. Purifications were performed on the 50 mL of Hi-5 insect cells cultures used for the YFP and CFP fluorescence kinetics (c). Deposits feature total lysate after freeze/thaw (T), supernatant after centrifugation (S), resin flow through (Ft) and the primary elution fraction (E). After migration on a 12% SDS-PAGE, proteins were transferred on PVDF membrane. Primary antibodies targeting human-IAV PB2 (rabbit IgG) and human-IAV PB1 (mouse IgG) have been used. Revelation was performed with secondary goat antibodies coupled with Alexa Fluor 532 (λexi = 632 nm, λemi = 647 nm) and Alexa Fluor 633 (λexi = 531 nm, λemi = 554 nm) targeting mouse and rabbit H + L domains respectively, using a Typhoon Trio imaging system (GE Healthcare). After integration of the raw data, PB1 and PB2 revelation are visible in red and green respectively. Black and white signal of PB2 is also shown (bottom) to highlight the PB2 truncations. The upper bands appearing in green/yellow, correspond to unprocessed polyproteins.
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f1: Truncated human-IAV RdRp polyprotein expression.(a) Logic of the TEV-PA-PB1-PB2-CFP polyprotein expression in a schematic view. During expression, TEV will process the polyprotein ensuring a stoichiometric assembly of PA, PB1 and PB2. YFP and CFP are produced during the process and monitor respectively baculovirus proliferation and polyprotein translation. (b) Truncated RdRp constructs where PB2 is incrementally extended until residues 116, 320 or 483. (c) YFP (left) and CFP (right) fluorescence kinetics measured during Hi-5 insect cells culture of truncated RdRp constructs. YFP (λexi = 488 nm, λemi = 525 nm) and CFP (λexi = 430 nm, λemi = 480 nm) measurements were performed on cellular extracts prepared by sonicating 1 × 106 cells in PBS (500 μL) follow by centrifugation. Fluorescence intensities are plotted against time after infection. (d) Small scale nickel resin purification analysis by western blot. Purifications were performed on the 50 mL of Hi-5 insect cells cultures used for the YFP and CFP fluorescence kinetics (c). Deposits feature total lysate after freeze/thaw (T), supernatant after centrifugation (S), resin flow through (Ft) and the primary elution fraction (E). After migration on a 12% SDS-PAGE, proteins were transferred on PVDF membrane. Primary antibodies targeting human-IAV PB2 (rabbit IgG) and human-IAV PB1 (mouse IgG) have been used. Revelation was performed with secondary goat antibodies coupled with Alexa Fluor 532 (λexi = 632 nm, λemi = 647 nm) and Alexa Fluor 633 (λexi = 531 nm, λemi = 554 nm) targeting mouse and rabbit H + L domains respectively, using a Typhoon Trio imaging system (GE Healthcare). After integration of the raw data, PB1 and PB2 revelation are visible in red and green respectively. Black and white signal of PB2 is also shown (bottom) to highlight the PB2 truncations. The upper bands appearing in green/yellow, correspond to unprocessed polyproteins.

Mentions: Polyproteins are naturally used by certain viruses to structure their proteome1415. Recombinant polyproteins mimicking viral polyproteins have recently emerged as a powerful tool to express protein complexes for functional characterisation as well as structural determination (reviewed in16). Using this strategy17, soluble and active heterotrimeric RNA-dependent RNA-polymerases of influenza B virus (IBV) and bat influenza A (bat-IAV) virus could be produced, crystallized and the structure determined at high resolution56. We have applied a similar strategy to the RdRp of two human-IAV strains, A/Victoria/3/1975(H3N2) and the highly pathogenic A/Viet-Nam/1203/2004(H5N1). The three genes of each heterotrimeric complex were combined with Tobacco Etch Virus (TEV) protease and Cyan Fluorescent Protein (CFP) in a single large open reading frame (ORF). Each gene was separated by a DNA sequence encoding for a peptide segment comprising a short serine/glycine linker and a TEV protease cleavage site (Fig. 1a and supplementary Figure 1). The constructs were optimized for recombinant expression in insect cells using the MultiBac system1819. During expression, the TEV protease cleaves the polyprotein co-translationally, resulting in a stoichiometric assembly of the RdRp that can then be selectively purified using an affinity nickel resin. The production of the polyprotein is monitored using the fluorescence of two reporter proteins: CFP encoded within the polyprotein reports directly on recombinant protein yield, while YFP, integrated in the baculovirus genome, monitors virus performance19. The ratio of YFP/CFP in our system is a highly useful criterion to determine recombinant polyprotein translation efficacy of the different constructs.


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)

Truncated human-IAV RdRp polyprotein expression.(a) Logic of the TEV-PA-PB1-PB2-CFP polyprotein expression in a schematic view. During expression, TEV will process the polyprotein ensuring a stoichiometric assembly of PA, PB1 and PB2. YFP and CFP are produced during the process and monitor respectively baculovirus proliferation and polyprotein translation. (b) Truncated RdRp constructs where PB2 is incrementally extended until residues 116, 320 or 483. (c) YFP (left) and CFP (right) fluorescence kinetics measured during Hi-5 insect cells culture of truncated RdRp constructs. YFP (λexi = 488 nm, λemi = 525 nm) and CFP (λexi = 430 nm, λemi = 480 nm) measurements were performed on cellular extracts prepared by sonicating 1 × 106 cells in PBS (500 μL) follow by centrifugation. Fluorescence intensities are plotted against time after infection. (d) Small scale nickel resin purification analysis by western blot. Purifications were performed on the 50 mL of Hi-5 insect cells cultures used for the YFP and CFP fluorescence kinetics (c). Deposits feature total lysate after freeze/thaw (T), supernatant after centrifugation (S), resin flow through (Ft) and the primary elution fraction (E). After migration on a 12% SDS-PAGE, proteins were transferred on PVDF membrane. Primary antibodies targeting human-IAV PB2 (rabbit IgG) and human-IAV PB1 (mouse IgG) have been used. Revelation was performed with secondary goat antibodies coupled with Alexa Fluor 532 (λexi = 632 nm, λemi = 647 nm) and Alexa Fluor 633 (λexi = 531 nm, λemi = 554 nm) targeting mouse and rabbit H + L domains respectively, using a Typhoon Trio imaging system (GE Healthcare). After integration of the raw data, PB1 and PB2 revelation are visible in red and green respectively. Black and white signal of PB2 is also shown (bottom) to highlight the PB2 truncations. The upper bands appearing in green/yellow, correspond to unprocessed polyproteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Truncated human-IAV RdRp polyprotein expression.(a) Logic of the TEV-PA-PB1-PB2-CFP polyprotein expression in a schematic view. During expression, TEV will process the polyprotein ensuring a stoichiometric assembly of PA, PB1 and PB2. YFP and CFP are produced during the process and monitor respectively baculovirus proliferation and polyprotein translation. (b) Truncated RdRp constructs where PB2 is incrementally extended until residues 116, 320 or 483. (c) YFP (left) and CFP (right) fluorescence kinetics measured during Hi-5 insect cells culture of truncated RdRp constructs. YFP (λexi = 488 nm, λemi = 525 nm) and CFP (λexi = 430 nm, λemi = 480 nm) measurements were performed on cellular extracts prepared by sonicating 1 × 106 cells in PBS (500 μL) follow by centrifugation. Fluorescence intensities are plotted against time after infection. (d) Small scale nickel resin purification analysis by western blot. Purifications were performed on the 50 mL of Hi-5 insect cells cultures used for the YFP and CFP fluorescence kinetics (c). Deposits feature total lysate after freeze/thaw (T), supernatant after centrifugation (S), resin flow through (Ft) and the primary elution fraction (E). After migration on a 12% SDS-PAGE, proteins were transferred on PVDF membrane. Primary antibodies targeting human-IAV PB2 (rabbit IgG) and human-IAV PB1 (mouse IgG) have been used. Revelation was performed with secondary goat antibodies coupled with Alexa Fluor 532 (λexi = 632 nm, λemi = 647 nm) and Alexa Fluor 633 (λexi = 531 nm, λemi = 554 nm) targeting mouse and rabbit H + L domains respectively, using a Typhoon Trio imaging system (GE Healthcare). After integration of the raw data, PB1 and PB2 revelation are visible in red and green respectively. Black and white signal of PB2 is also shown (bottom) to highlight the PB2 truncations. The upper bands appearing in green/yellow, correspond to unprocessed polyproteins.
Mentions: Polyproteins are naturally used by certain viruses to structure their proteome1415. Recombinant polyproteins mimicking viral polyproteins have recently emerged as a powerful tool to express protein complexes for functional characterisation as well as structural determination (reviewed in16). Using this strategy17, soluble and active heterotrimeric RNA-dependent RNA-polymerases of influenza B virus (IBV) and bat influenza A (bat-IAV) virus could be produced, crystallized and the structure determined at high resolution56. We have applied a similar strategy to the RdRp of two human-IAV strains, A/Victoria/3/1975(H3N2) and the highly pathogenic A/Viet-Nam/1203/2004(H5N1). The three genes of each heterotrimeric complex were combined with Tobacco Etch Virus (TEV) protease and Cyan Fluorescent Protein (CFP) in a single large open reading frame (ORF). Each gene was separated by a DNA sequence encoding for a peptide segment comprising a short serine/glycine linker and a TEV protease cleavage site (Fig. 1a and supplementary Figure 1). The constructs were optimized for recombinant expression in insect cells using the MultiBac system1819. During expression, the TEV protease cleaves the polyprotein co-translationally, resulting in a stoichiometric assembly of the RdRp that can then be selectively purified using an affinity nickel resin. The production of the polyprotein is monitored using the fluorescence of two reporter proteins: CFP encoded within the polyprotein reports directly on recombinant protein yield, while YFP, integrated in the baculovirus genome, monitors virus performance19. The ratio of YFP/CFP in our system is a highly useful criterion to determine recombinant polyprotein translation efficacy of the different constructs.

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