I222 Neuraminidase Mutations Further Reduce Oseltamivir Susceptibility of Indonesian Clade 2.1 Highly Pathogenic Avian Influenza A(H5N1) Viruses

McKimm-Breschkin, Jennifer L.; Barrett, Susan; Pudjiatmoko,; Azhar, Muhammad; Wong, Frank; Selleck, Paul; Mohr, Peter G.; McGrane, James; Kim, Mia

doi:10.1371/journal.pone.0066105

Cited by 28 publications

(34 citation statements)

References 52 publications

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“…This residue may indirectly affect the enzyme active site via hydrogen bonding with the residue at position 274 (44), which may also explain the importance of this residue in sensitivity of the NA protein toward oseltamivir carboxylate (44-46). H5N1 viruses with an H at position 252 of the NA protein were shown to be more susceptible to oseltamivir carboxylate than viruses with a Y at this position (47)(48)(49)(50). Similar results were obtained when our recombinant N1 proteins were analyzed for oseltamivir sensitivity (data not shown).…”

Section: Discussionsupporting

confidence: 76%

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

Dai

Guo

Dortmans

et al. 2016

J Virol

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Influenza A virus (IAV) attachment to and release from sialoside receptors is determined by the balance between hemagglutinin (HA) and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the K m value but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled-coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95) and therefore the specific activity or sialic acid binding affinity (K m value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity. IMPORTANCEThe IAV HA and NA glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities, as they are easily purified from cell culture media and applied in downstream assays. In the present study, we analyzed the enzymatic activity of different NA ectodomains with three commonly used tetramerization domains and compared them with fulllength NA proteins. By performing a mutagenic analysis, we identified several residues that affected NA assembly, activity, and/or substrate binding. In addition, our results indicate that the design of the recombinant soluble NA protein, including the particular tetramerization domain, is an important determinant for maintaining the enzymatic properties within the head domain. NA ectodomains extended with a Tetrabrachion domain better mimicked the full-length proteins than when the other tetramerization domains were used. V irus particles contain dedicated proteins that recognize cell surface molecules. While some viruses evolved to bind specific protein receptors, others bind carbohydrate moieties, such as sialic acids (SIAs), that are omnipresent on the cell surface as well as in the mucus. Several of the latter viruses, including influenza A virus (IAV), carry receptor-destroying activities in addit...

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Section: Discussionsupporting

confidence: 76%

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

Dai

Guo

Dortmans

et al. 2016

J Virol

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“…H5N1 clade 1 A/Chicken/Vietnam/08/2004 and clade 2 A/Chicken/Bangli/BBVD-563/2007, the latter with decreased sensitivity to oseltamivir, (McKimm-Breschkin et al, 2013a) were grown in eggs at CSIRO AAHL and the allantoic fluid was gamma irradiated as previously described (McKimm-Breschkin et al, 2013a). …”

Section: Methodsmentioning

confidence: 99%

“…Each inhibitor, at a concentration of approximately 50 times the IC 50 (Barrett et al, 2011; McKimm-Breschkin et al, 2013a) was diluted in PBS and was added to three or four virus coated wells. PBS was added to two or three wells for the untreated virus controls.…”

Section: Methodsmentioning

confidence: 99%

“…For slow binding NAIs pre-incubation enhances binding, leading to lower IC 50s compared to the no preincubation reaction. While the NAIs bind slowly to wild type viruses, we saw a loss of slow binding with viruses with NA mutations conferring reduced susceptibility (Barrett et al, 2011; McKimm-Breschkin et al, 2013a; McKimm-Breschkin et al, 2013b; Oakley et al, 2010). …”

Section: Introductionmentioning

confidence: 91%

“…Many mutations which lead to NAI resistance, also lead to loss of slow binding of the NAI (Barrett et al, 2011; Baum et al, 2003; Blick et al, 1995; McKimm-Breschkin et al, 1998; Oakley et al, 2010). We have recently developed a simple phenotypic assay which allows the easy identification of slow and fast binding of NAIs and multiple viruses without requiring purified virus or NA, or a detailed knowledge of enzyme kinetics (Barrett et al, 2011; McKimm-Breschkin et al, 2013a; McKimm-Breschkin et al, 2012; McKimm-Breschkin et al, 2013b; Oakley et al, 2010). The analysis uses two assays, where we follow the changes in IC 50 , each 10 minutes for 60 minutes after addition of substrate.…”

Section: Introductionmentioning

confidence: 99%

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Solid phase assay for comparing reactivation rates of neuraminidases of influenza wild type and resistant mutants after inhibitor removal

Barrett

McKimm-Breschkin

2014

Antiviral Research

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The influenza virus neuraminidase inhibitors are normally slow binding inhibitors, but many mutations leading to resistance, also result in the loss of the slow binding phenotype. Mutations can also affect the rate of dissociation of the inhibitors from the neuraminidase, but the assays to measure this require large amounts of virus and are time consuming. To more fully understand the impacts of mutations on the binding and dissociation of the neuraminidase inhibitors we have developed a solid phase reactivation assay, which can use small amounts of crude virus sample bound to an ELISA plate. Multiple viruses can be assayed simultaneously against multiple inhibitors. Using this assay we have demonstrated differences in the relative rates of dissociation of the inhibitors and reactivation of enzyme activity among different influenza A and B viruses for zanamivir, oseltamivir and peramivir. In general oseltamivir dissociated the fastest, and dissociation of peramivir was much slower than both the other inhibitors. Viruses with H274Y, E119V and E119G mutations demonstrated faster dissociation of the inhibitor to which they were resistant. Dissociation of zanamivir and oseltamivir were faster from the D197E mutant, but not of peramivir.

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H5N1 Highly Pathogenic Avian Influenza in Indonesia: Retrospective Considerations

Daniels

Wiyono

Sawitri

et al. 2012

Current Topics in Microbiology and Immunology

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Indonesia is one of the five countries where highly pathogenic avian influenza viruses of the H5N1 subtype (H5N1 HPAI) remain endemic in poultry. Importantly, it is one of the countries where the virus causes human infections. WHO data indicate that as of 2 May 2012, 189 human cases of Influenza A (H5N1) had been reported in Indonesia, with 157 human deaths. These human cases included a small number in which limited human-to-human transmission could have occurred. Hence, there remains a critical need in Indonesia for a more effective One Health approach to the control and prevention of this disease in people and in poultry. This chapter explores a number of aspects of the evolution of this disease in Indonesia, the virus that causes it and the control and preventive measures introduced, focusing on the successes and shortcomings of veterinary and One Health approaches. Indonesia provides many examples of situations where this latter approach has been successful, and others where further work is needed to maximize the benefits from coordinated responses to this disease leading to effective management of the risk to human health.

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I222 Neuraminidase Mutations Further Reduce Oseltamivir Susceptibility of Indonesian Clade 2.1 Highly Pathogenic Avian Influenza A(H5N1) Viruses

Cited by 28 publications

References 52 publications

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

Solid phase assay for comparing reactivation rates of neuraminidases of influenza wild type and resistant mutants after inhibitor removal

H5N1 Highly Pathogenic Avian Influenza in Indonesia: Retrospective Considerations

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