Limits...
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.

Show MeSH

Related in: MedlinePlus

The RNA-binding interface within the LASV NP RNase domain.A, the dsRNA substrate bound in the surface cleft is shown as a stick model. The cleaving strand of dsRNA is shown in magenta and the complementary strand in cyan. B, schematic representation of the 4-bp dsRNA substrate and its interacting residues within the LASV NP RNase domain. The scissile bond is indicated with a scissor. Mn2+ is shown as a black ball.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3675627&req=5

Figure 3: The RNA-binding interface within the LASV NP RNase domain.A, the dsRNA substrate bound in the surface cleft is shown as a stick model. The cleaving strand of dsRNA is shown in magenta and the complementary strand in cyan. B, schematic representation of the 4-bp dsRNA substrate and its interacting residues within the LASV NP RNase domain. The scissile bond is indicated with a scissor. Mn2+ is shown as a black ball.

Mentions: Electron density of the RNA substrate clearly showed the presence of 4 bp (5′-GGGC-3′/3′-CCCG-5′) in the catalytic cavity (Fig. 2C). Similarly, the 3′-5′ exonucleases Trex1 and Trex2 also contain 4 bases of their ssDNA substrates in the catalytic pockets (20–22). The dsRNA substrate in the LASV NP-C catalytic site corresponds to the first 4 bp (5′-GGGC-3′/3′-CCCG-5′) of the input 8-bp dsRNA (5′-GGGCGCCC-3′/3′-CCCGCGGG-5′. For convenience, the 8-bp RNA is numbered as 5′-G1G2G3C4G5C6C7C8–3′ (Fig. 3A). This was because a catalytically active NP-C domain was used in the co-crystallization process and was predicted to have removed the last 4 nucleotides from the 3′ end of the cleaving strand, resulting in an intermediate dsRNA product with a 5′ protruding end of the complementary strand (5′-GGGC-3′/3′-CCCGCGGG-5′). The 4 nucleotides 5′-GGGC-3′ of the cleaving strand were almost completely buried in the catalytic cavity (Fig. 3A). Residues Asp-426, Glu-391, Asp-466, Gln-462, and Lys-488 in the cavity interacted directly with the cleaving stand, whereas other residues in the cavity coordinated the binding indirectly through water molecules (Fig. 3, A and B). Most of the residues interacting with the cleaving strand are phylogenetically conserved among arenaviruses, such as Gly-392, His-431, Glu-391, Asp-466, Gln-462, Arg-492, and Ser-430. In particular, the 3′-end ribonucleotide C at position 4 of the cleaving strand was coordinately anchored by several residues: Arg-492 through a water molecule forming a hydrogen bridge with the phosphate group, and Arg-426 and Glu-391 through their side chains forming a hydrogen bond with the 2′- and 3′-oxygen of the ribose of the cytosine ribonucleoside, respectively (Fig. 3B). Gln-462 and Asp-466 bound the guanine ribonucleotide (G) at the 3 position through the side chains forming a hydrogen bond with the phosphoryl oxygen and 2′-oxygen of the ribose (Fig. 3B). Lys-488 formed two salt bridges with the phosphates of the two Gs at the 3 and 2 positions, respectively (Fig. 3B). Meanwhile, the 3′-CCCG-5′ nucleotides of the complementary strand were stacked against the aromatic side chain of Tyr-429 and were anchored by the side chains of residues Arg-393, Gln-425, Asp-426, Gln-422, and Asp-465, whereas the rest of the nucleotides of the complementary strand (3′-CGGG-5′) were exposed to solvent (Fig. 3B). Interestingly, residues involved in the complementary strand binding are not phylogenetically conserved.


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)

The RNA-binding interface within the LASV NP RNase domain.A, the dsRNA substrate bound in the surface cleft is shown as a stick model. The cleaving strand of dsRNA is shown in magenta and the complementary strand in cyan. B, schematic representation of the 4-bp dsRNA substrate and its interacting residues within the LASV NP RNase domain. The scissile bond is indicated with a scissor. Mn2+ is shown as a black ball.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675627&req=5

Figure 3: The RNA-binding interface within the LASV NP RNase domain.A, the dsRNA substrate bound in the surface cleft is shown as a stick model. The cleaving strand of dsRNA is shown in magenta and the complementary strand in cyan. B, schematic representation of the 4-bp dsRNA substrate and its interacting residues within the LASV NP RNase domain. The scissile bond is indicated with a scissor. Mn2+ is shown as a black ball.
Mentions: Electron density of the RNA substrate clearly showed the presence of 4 bp (5′-GGGC-3′/3′-CCCG-5′) in the catalytic cavity (Fig. 2C). Similarly, the 3′-5′ exonucleases Trex1 and Trex2 also contain 4 bases of their ssDNA substrates in the catalytic pockets (20–22). The dsRNA substrate in the LASV NP-C catalytic site corresponds to the first 4 bp (5′-GGGC-3′/3′-CCCG-5′) of the input 8-bp dsRNA (5′-GGGCGCCC-3′/3′-CCCGCGGG-5′. For convenience, the 8-bp RNA is numbered as 5′-G1G2G3C4G5C6C7C8–3′ (Fig. 3A). This was because a catalytically active NP-C domain was used in the co-crystallization process and was predicted to have removed the last 4 nucleotides from the 3′ end of the cleaving strand, resulting in an intermediate dsRNA product with a 5′ protruding end of the complementary strand (5′-GGGC-3′/3′-CCCGCGGG-5′). The 4 nucleotides 5′-GGGC-3′ of the cleaving strand were almost completely buried in the catalytic cavity (Fig. 3A). Residues Asp-426, Glu-391, Asp-466, Gln-462, and Lys-488 in the cavity interacted directly with the cleaving stand, whereas other residues in the cavity coordinated the binding indirectly through water molecules (Fig. 3, A and B). Most of the residues interacting with the cleaving strand are phylogenetically conserved among arenaviruses, such as Gly-392, His-431, Glu-391, Asp-466, Gln-462, Arg-492, and Ser-430. In particular, the 3′-end ribonucleotide C at position 4 of the cleaving strand was coordinately anchored by several residues: Arg-492 through a water molecule forming a hydrogen bridge with the phosphate group, and Arg-426 and Glu-391 through their side chains forming a hydrogen bond with the 2′- and 3′-oxygen of the ribose of the cytosine ribonucleoside, respectively (Fig. 3B). Gln-462 and Asp-466 bound the guanine ribonucleotide (G) at the 3 position through the side chains forming a hydrogen bond with the phosphoryl oxygen and 2′-oxygen of the ribose (Fig. 3B). Lys-488 formed two salt bridges with the phosphates of the two Gs at the 3 and 2 positions, respectively (Fig. 3B). Meanwhile, the 3′-CCCG-5′ nucleotides of the complementary strand were stacked against the aromatic side chain of Tyr-429 and were anchored by the side chains of residues Arg-393, Gln-425, Asp-426, Gln-422, and Asp-465, whereas the rest of the nucleotides of the complementary strand (3′-CGGG-5′) were exposed to solvent (Fig. 3B). Interestingly, residues involved in the complementary strand binding are not phylogenetically conserved.

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