Abstract
The influenza surface glycoprotein neuraminidase (NA) is essential for the efficient spread of the virus. Antiviral drugs such as Tamiflu (oseltamivir) and Relenza (zanamivir) that inhibit NA enzyme activity have been shown to be effective in the treatment of influenza infections. The recent ‘swine flu’ pandemic and world-wide emergence of Tamiflu-resistant seasonal human influenza A(H1N1) H274Y have highlighted the need for the ongoing development of new anti-virals, efficient production of vaccine proteins and novel diagnostic tools. Each of these goals could benefit from the production of large quantities of highly pure and stable NA. This publication describes a generic expression system for NAs in a baculovirus Expression Vector System (BEVS) that is capable of expressing milligram amounts of recombinant NA. To construct NAs with increased stability, the natural influenza NA stalk was replaced by two different artificial tetramerization domains that drive the formation of catalytically active NA homotetramers: GCN4-pLI from yeast or the Tetrabrachion tetramerization domain from Staphylothermus marinus. Both recombinant NAs are secreted as FLAG-tagged proteins to allow for rapid and simple purification. The Tetrabrachion-based NA showed good solubility, increased stability and biochemical properties closer to the original viral NA than the GCN4-pLI based construct. The expressed quantities and high quality of the purified recombinant NA suggest that this expression system is capable of producing recombinant NA for a broad range of applications including high-throughput drug screening, protein crystallisation, or vaccine development.
Highlights
Human seasonal influenza is responsible for a world-wide death toll of an estimated 250,000–500,000 and 3–5 million cases of severe illness routinely every year (WHO influenza fact sheet 2009), a fact forgotten in light of the media presence during the rapid spread of the ‘swine flu’ virus (human A(H1N1)-2009), the first influenza pandemic in more than 4 decades
Construction of a generic expression system In order to allow the expression of a broad range of NAs and to identify conserved regions in the NA stalk domain, a multisequence alignment of 43 NA sequences ranging from N1 to N9 (1918–2007) was performed
Characterizing and optimizing the expression system Sf21 cells were infected with all 4 constructs at a Multiplicity of infection (MOI) of 1, 2, 3, and 4 and the respective NA activity in the media was measured after 0 h, 24 h, 48 h, and 72 h
Summary
Human seasonal influenza is responsible for a world-wide death toll of an estimated 250,000–500,000 and 3–5 million cases of severe illness routinely every year (WHO influenza fact sheet 2009), a fact forgotten in light of the media presence during the rapid spread of the ‘swine flu’ virus (human A(H1N1)-2009), the first influenza pandemic in more than 4 decades. One of the major influenza proteins that represents an important component of killed split vaccines is the viral neuraminidase (NA). NA inhibitor-resistant virus strains have recently emerged such as the A(H1N1) seasonal human influenza strain with an H274Y mutation, which confers resistance to Tamiflu [3,4,5]. Understanding the mechanisms behind antiviral resistance and the continual development of new antivirals based on that insight is critically important. Protein crystallisation has been a powerful approach to explain mechanisms of resistance, the production of large amounts of highly purified NA has been problematic. Often mutations that confer resistance have been shown to compromise virus viability resulting in extremely low levels of purified NA being obtained[8,9]. Viral NAs can become intrinsically unstable once they are cleaved from the membrane[11] and lose activity within hours and impede any subsequent analysis
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