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Structures of arenaviral nucleoproteins with triphosphate dsRNA reveal a unique mechanism of immune suppression.

Jiang X, Huang Q, Wang W, Dong H, Ly H, Liang Y, Dong C - J. Biol. Chem. (2013)

Bottom Line: How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown.We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5' triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production.New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

View Article: PubMed Central - PubMed

Affiliation: Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3'-5' exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5' triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

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Time course structural analysis of LASV NP-C·dsRNA complex soaked in Mn2+ solution.Fo − Fc electron density map of the 5′-ppp dsRNA is shown in blue contoured at 2.5 σ, and the 2Fo − Fc electron density map of the active site residues are shown in orange contoured at 1 σ. A, only water molecules, but not Mn2+, are present in the active site prior to the soaking. B, after soaking in Mn2+ solution for 1 min, one Mn2+ was identified and was coordinated by three water molecules, side chain (OD1) of Asp-389 and two phosphoryl oxygen molecules (O1P and O3*) of the dsRNA substrate (supplemental Fig. S4). The electron density for the 5′-ppp moiety (triphosphate) of the cleaving could be detected. C, after soaking for 1.5 min, only one Mn2+ ion can be seen with three coordinating water molecules. D, after soaking in Mn2+ solution for 5 min, the electron density of the 1st Mn2+ appeared, and the electron density of the side chain of Tyr-429 disappeared, whereas other local residues did not change, suggesting that Tyr-429 became structurally disordered.
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Figure 6: Time course structural analysis of LASV NP-C·dsRNA complex soaked in Mn2+ solution.Fo − Fc electron density map of the 5′-ppp dsRNA is shown in blue contoured at 2.5 σ, and the 2Fo − Fc electron density map of the active site residues are shown in orange contoured at 1 σ. A, only water molecules, but not Mn2+, are present in the active site prior to the soaking. B, after soaking in Mn2+ solution for 1 min, one Mn2+ was identified and was coordinated by three water molecules, side chain (OD1) of Asp-389 and two phosphoryl oxygen molecules (O1P and O3*) of the dsRNA substrate (supplemental Fig. S4). The electron density for the 5′-ppp moiety (triphosphate) of the cleaving could be detected. C, after soaking for 1.5 min, only one Mn2+ ion can be seen with three coordinating water molecules. D, after soaking in Mn2+ solution for 5 min, the electron density of the 1st Mn2+ appeared, and the electron density of the side chain of Tyr-429 disappeared, whereas other local residues did not change, suggesting that Tyr-429 became structurally disordered.

Mentions: Although DEDDH nucleases are known to cleave ssRNA or DNA via the two-metal mechanism (25, 26), how LASV NP, a 3–5′ DEDDH exoribonuclease, degrades dsRNA is not clear. Structures of only a few known 3′-5′ exonucleases, such as Trex1, Trex2, and Klenow fragment, in complex with their DNA substrates are available (20, 22, 23), each using a catalytically inactive protein. The NP-C·dsRNA complex structure in this study contains an enzymatically active exoribonuclease domain of LASV NP and a dsRNA substrate in the catalytic pocket. This provides a unique opportunity to investigate the kinetic process of how this particular 3′-5′ exoribonuclease activity degrades dsRNA substrate. To do this, we set up a time course experiment to capture the conformational changes of the NP-C·dsRNA complex during the enzymatic reaction. The LASV NP-C·dsRNA complex was soaked in a cryoprotectant solution with 100 mm MnCl2, and the crystals were incubated for 1, 1.5, 5, or 10 min at room temperature under a standard crystallization condition but with pH 5.5 that would slow down the catalytic reaction for the purpose of capturing the different stages of the reaction (Fig. 6). Crystals that were soaked for 10 min were not good for data collection. Over the time course, the crystal cell volumes changed gradually from 474,825 Å3 to 444,643 Å3 (Table 1). Several changes were noted. First of all, the electron density signal of the 1st Mn2+ ion, which was visualized in the RNA-free Mn2+-bound NP structure (PDB code 3MWT), was not clear at first in the co-crystal structure but became visible over time. In contrast, the 2nd Mn2+ ion, which was not visible in the RNA-free NP structure (PDB code 3MWT), was now clearly visible and was coordinated by the side chain (OD1) of Asp-389, two scissile phosphoryl oxygen molecules (O1P and O3*) of the dsRNA substrate, and three water molecules (Fig. 6 and supplemental S4 and S5). Second, the electron density of the 5′-ppp moiety of the triphosphate dsRNA substrate became clearly visible, and that of an additional nucleotide C4 at the 5′ end of the complementary strand also became partially visible. This strongly suggests that the RNA molecule identified in the NP-C·dsRNA complex structure (Fig. 3) is indeed an intermediate product of the NP-C exoribonuclease activity. In addition, the electron density for the 4-bp dsRNA in the catalytic cavity became less clear over time, which serves as evidence for the dsRNA moving toward the catalytically active site during the cleavage reaction (Fig. 6C and supplemental Fig. S3). Last, the side chain of residue Tyr-429 became structurally disordered, whereas those of other local residues remained unchanged (Fig. 6D), indicating that the Tyr-429 residue moved from the G5 base to stack against the next C6 base of the complementary strand in the 5′ to 3′ direction (3′-CCCGXXXX-5′) when the cytidine monophosphate was removed from the 3′ end of the cleaving strand (5′-GGGC-3′).


Structures of arenaviral nucleoproteins with triphosphate dsRNA reveal a unique mechanism of immune suppression.

Jiang X, Huang Q, Wang W, Dong H, Ly H, Liang Y, Dong C - J. Biol. Chem. (2013)

Time course structural analysis of LASV NP-C·dsRNA complex soaked in Mn2+ solution.Fo − Fc electron density map of the 5′-ppp dsRNA is shown in blue contoured at 2.5 σ, and the 2Fo − Fc electron density map of the active site residues are shown in orange contoured at 1 σ. A, only water molecules, but not Mn2+, are present in the active site prior to the soaking. B, after soaking in Mn2+ solution for 1 min, one Mn2+ was identified and was coordinated by three water molecules, side chain (OD1) of Asp-389 and two phosphoryl oxygen molecules (O1P and O3*) of the dsRNA substrate (supplemental Fig. S4). The electron density for the 5′-ppp moiety (triphosphate) of the cleaving could be detected. C, after soaking for 1.5 min, only one Mn2+ ion can be seen with three coordinating water molecules. D, after soaking in Mn2+ solution for 5 min, the electron density of the 1st Mn2+ appeared, and the electron density of the side chain of Tyr-429 disappeared, whereas other local residues did not change, suggesting that Tyr-429 became structurally disordered.
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Figure 6: Time course structural analysis of LASV NP-C·dsRNA complex soaked in Mn2+ solution.Fo − Fc electron density map of the 5′-ppp dsRNA is shown in blue contoured at 2.5 σ, and the 2Fo − Fc electron density map of the active site residues are shown in orange contoured at 1 σ. A, only water molecules, but not Mn2+, are present in the active site prior to the soaking. B, after soaking in Mn2+ solution for 1 min, one Mn2+ was identified and was coordinated by three water molecules, side chain (OD1) of Asp-389 and two phosphoryl oxygen molecules (O1P and O3*) of the dsRNA substrate (supplemental Fig. S4). The electron density for the 5′-ppp moiety (triphosphate) of the cleaving could be detected. C, after soaking for 1.5 min, only one Mn2+ ion can be seen with three coordinating water molecules. D, after soaking in Mn2+ solution for 5 min, the electron density of the 1st Mn2+ appeared, and the electron density of the side chain of Tyr-429 disappeared, whereas other local residues did not change, suggesting that Tyr-429 became structurally disordered.
Mentions: Although DEDDH nucleases are known to cleave ssRNA or DNA via the two-metal mechanism (25, 26), how LASV NP, a 3–5′ DEDDH exoribonuclease, degrades dsRNA is not clear. Structures of only a few known 3′-5′ exonucleases, such as Trex1, Trex2, and Klenow fragment, in complex with their DNA substrates are available (20, 22, 23), each using a catalytically inactive protein. The NP-C·dsRNA complex structure in this study contains an enzymatically active exoribonuclease domain of LASV NP and a dsRNA substrate in the catalytic pocket. This provides a unique opportunity to investigate the kinetic process of how this particular 3′-5′ exoribonuclease activity degrades dsRNA substrate. To do this, we set up a time course experiment to capture the conformational changes of the NP-C·dsRNA complex during the enzymatic reaction. The LASV NP-C·dsRNA complex was soaked in a cryoprotectant solution with 100 mm MnCl2, and the crystals were incubated for 1, 1.5, 5, or 10 min at room temperature under a standard crystallization condition but with pH 5.5 that would slow down the catalytic reaction for the purpose of capturing the different stages of the reaction (Fig. 6). Crystals that were soaked for 10 min were not good for data collection. Over the time course, the crystal cell volumes changed gradually from 474,825 Å3 to 444,643 Å3 (Table 1). Several changes were noted. First of all, the electron density signal of the 1st Mn2+ ion, which was visualized in the RNA-free Mn2+-bound NP structure (PDB code 3MWT), was not clear at first in the co-crystal structure but became visible over time. In contrast, the 2nd Mn2+ ion, which was not visible in the RNA-free NP structure (PDB code 3MWT), was now clearly visible and was coordinated by the side chain (OD1) of Asp-389, two scissile phosphoryl oxygen molecules (O1P and O3*) of the dsRNA substrate, and three water molecules (Fig. 6 and supplemental S4 and S5). Second, the electron density of the 5′-ppp moiety of the triphosphate dsRNA substrate became clearly visible, and that of an additional nucleotide C4 at the 5′ end of the complementary strand also became partially visible. This strongly suggests that the RNA molecule identified in the NP-C·dsRNA complex structure (Fig. 3) is indeed an intermediate product of the NP-C exoribonuclease activity. In addition, the electron density for the 4-bp dsRNA in the catalytic cavity became less clear over time, which serves as evidence for the dsRNA moving toward the catalytically active site during the cleavage reaction (Fig. 6C and supplemental Fig. S3). Last, the side chain of residue Tyr-429 became structurally disordered, whereas those of other local residues remained unchanged (Fig. 6D), indicating that the Tyr-429 residue moved from the G5 base to stack against the next C6 base of the complementary strand in the 5′ to 3′ direction (3′-CCCGXXXX-5′) when the cytidine monophosphate was removed from the 3′ end of the cleaving strand (5′-GGGC-3′).

Bottom Line: How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown.We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5' triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production.New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

View Article: PubMed Central - PubMed

Affiliation: Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3'-5' exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5' triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

Show MeSH
Related in: MedlinePlus