Nucleoside Monophosphate Complex Structures of the Endonuclease Domain from the Influenza Virus Polymerase PA Subunit Reveal the Substrate Binding Site inside the Catalytic Center

Zhao, Cong; Lou, Zhiyong; Guo, Yu; Ma, Ming; Chen, Yutao; Liang, Shuaiyi; Zhang, Liang; Chen, Shoudeng; Li, Xuemei; Liu, Yingfang; Bartlam, Mark; Rao, Zihe

doi:10.1128/jvi.00911-09

Cited by 56 publications

(91 citation statements)

References 23 publications

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“…This sulfate is in the same position as one of the phosphates of the EcoRV restriction enzyme product complex (16). Tyr130 and Lys137 coordinate water molecules that bind to the monophosphate group in a complex of PA-Nter with nucleoside monophosphates (38). In the isolated endonuclease domain, the enzymatic activities of R84A, Y130A, and K137A were reduced, whereas in the context of the intact trimeric polymerase, the mutations retain full transcription activity (13).…”

Section: Discussionmentioning

confidence: 97%

“…The nuclease activity of PA-Nter and the mutants is higher in the presence of 1 mM MnCl 2 than in the presence of 1 mM MgCl 2 , and the shift in T m is higher in the presence of 0.1 mM Mn 2ϩ ions than in the presence of 5 mM Mg 2ϩ ions. Yuan and coworkers grew crystals of PA-Nter in the presence of 100 mM MgCl 2 and observed a single metal ion only in position M1, even in the presence of mononucleotide phosphates (38). Therefore, in the absence of substrate, Mg 2ϩ ions seem to bind only to the M1 site even at concentrations that lay several orders of magnitude above the low affinity K d (4 mM).…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, Yuan and coworkers crystallized PA-Nter in the presence of only MgCl 2 and observed a single Mg 2ϩ ion in site M1 (37). The active site also contains Lys134, which could correspond to the catalytic lysine of the PD-(D/E)XK motif (6,37,38).…”

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confidence: 99%

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Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit

Crépin

Dias

Palencia

et al. 2010

J Virol

136

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Influenza virus polymerase initiates the biosynthesis of its own mRNAs with capped 10-to 13-nucleotide fragments cleaved from cellular (pre-)mRNAs. Two activities are required for this cap-snatching activity: specific binding of the cap structure and an endonuclease activity. Recent work has shown that the cap-binding site is situated in the central part of the PB2 subunit and that the endonuclease activity is situated in the N-terminal domain of the PA subunit (PA-Nter). The influenza endonuclease is a member of the PD-(D/E)XK family of nucleases that use divalent metal ions for nucleic acid cleavage. Here we analyze the metal binding and endonuclease activities of eight PA-Nter single-point mutants. We show by calorimetry that the wild-type active site binds two Mn 2؉ ions and has a 500-fold higher affinity for manganese than for magnesium ions. The endonuclease activity of the isolated mutant domains are compared with the cap-dependent transcription activities of identical mutations in trimeric recombinant polymerases previously described by other groups. Mutations that inactivate the endonuclease activity in the isolated PA-Nter knock out the transcription but not replication activity in the recombinant polymerase. We confirm the importance of a number of active-site residues and identify some residues that may be involved in the positioning of the RNA substrate in the active site. Our results validate the use of the isolated endonuclease domain in a drug-design process for new anti-influenza virus compounds.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

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Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit

Crépin

Dias

Palencia

et al. 2010

J Virol

136

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“…1A), which are conserved among all PAn molecules from different strains, several other amino acids proposed to be important for interaction with the substrate (30) are conserved as well, namely, Ala17, Ala37, Ile38, Tyr130, and Lys137. Apparently, the amino acid substitutions A20T, L42M, C39S, and R196K in H17N10 PAn are not detrimental for its endonuclease activity, although these residues are conserved among the PAn molecules from the four influenza A viruses that have been characterized thus far and were proposed to be involved in substrate binding (30).…”

Section: Discussionmentioning

confidence: 99%

“…The H41A mutant was inactivated, showing an essential role for the histidine in this enzyme, as described for H5N1 PAn (16,30). This histidine residue is in vivo very likely to contribute to binding to both the ribose and the nucleoside of the substrate RNA (30).…”

Section: Discussionmentioning

confidence: 99%

The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity

Tefsen

Zhu

et al. 2014

J Virol

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e Influenza imposes a great burden on society, not only in its seasonal appearance that affects both humans and domesticated animals but also through the constant threat of potential pandemics. Migratory birds are considered to be the reservoir hosts for influenza viruses, but other animals must also be considered. The recently identified influenza-like virus genome, from H17N10 in bats, was shown to be markedly different from genomes of other known influenza viruses, as both its surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) do not have canonical functions. However, no studies on other individual proteins from this particular virus have been reported until now. Here, we describe the structure of the N-terminal domain of PA from H17N10 influenza-like virus at 2.7-Å resolution and show that it has a fold similar to those of homologous PA domains present in more familiar influenza A virus strains. Moreover, we demonstrate that it possesses endonuclease activity and that the histidine residue in the active site is essential for this activity. Although this particular influenza virus subtype is probably not infectious for humans (even its virus state has not been confirmed in bats, as only the genome has been sequenced), reassortment of canonical influenza viruses with certain segments from H17N10 cannot be ruled out at this stage. Therefore, further studies are urgently needed for the sake of influenza prevention and control.

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Structure and function of the influenza virus replication machinery and PB1‐F2

Mehle

McCullers

2013

Textbook of Influenza

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Nucleoside Monophosphate Complex Structures of the Endonuclease Domain from the Influenza Virus Polymerase PA Subunit Reveal the Substrate Binding Site inside the Catalytic Center

Cited by 56 publications

References 23 publications

Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit

Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit

The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity

Structure and function of the influenza virus replication machinery and PB1‐F2

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