Enhanced expression of secretable influenza virus neuraminidase in suspension mammalian cells by influenza virus nonstructural protein 1

Abstract

Influenza neuraminidase (NA) is a major target for anti-influenza drugs. With an increasing number of viruses resistant to the anti-NA drug oseltamivir, functionally active recombinant NA is needed for screening novel anti-NA compounds. In this study, the secretable NA (sNA) head domain of influenza A/Vietnam/DT-036/05 (H5N1) virus was expressed successfully in human embryonic kidney (HEK-293T) cells and shown to be enzymatically active. The inclusion of a plasmid encoding nonstructural protein 1 (NS1) of influenza A/Puerto Rico/8/34 virus with the sNA plasmid in the cotransfection demonstrated an increase in H5N1 sNA expression by 7.4 fold. Subsequently, the sNA/NS1 cotransfection protocol in serum-free 293-F suspension cell culture was optimized to develop a rapid transient gene expression (TGE) system for expression of large amounts of H5N1 sNA. Under optimized conditions, NS1 enhanced H5N1 sNA expression by 4.2 fold. The resulting H5N1 sNA displayed comparable molecular weight, glycosylation, K_m for MUNANA, and K_i for oseltamivir carboxylate to those of H5N1 NA on the virus surface. Taken together, the NS1-enhancing sNA expression strategy presented in this study could be used for rapid high-level expression of enzymatically active H5N1 sNA in suspension mammalian cells. This strategy may be applied for expression of sNA of other strains of influenza virus as well as the other recombinant proteins.

Introduction

Influenza neuraminidase (NA) is a viral surface glycoprotein whose role is to cleave sialic acids of host glycoproteins to allow the release of newly formed virions from infected cells (Compans et al., 1969, Dowdle et al., 1974). Although the neuraminidase inhibitors oseltamivir and zanamivir are effective against most strains of influenza A and B viruses (Kim et al., 1997, von Itzstein et al., 1993), oseltamivir-resistant variants continue to emerge (Kiso et al., 2010, Tamura et al., 2009). In 2005, for example, oseltamivir-resistant variants of avian influenza A (H5N1) containing NA mutations were isolated from patients (de Jong et al., 2005). In addition, global spread of the 2009 pandemic H1N1 influenza virus was also followed shortly by the emergence of oseltamivir-resistant variants (Le et al., 2010, Leung et al., 2009). These observations indicate an urgent need for new anti-NA compounds for controlling virus infection.

Screening for NA inhibitors requires a large amount of enzymatically active NA protein. Although native NA can be extracted directly from viral particles (McKimm-Breschkin et al., 1991, Wanitchang et al., 2010, Wu et al., 1995), recombinant NA expressed in cell cultures provides a safer alternative that does not rely on the limited supply of embryonated chicken eggs. So far, heterologous expression of NA has been reported in yeast, insect cells, and mammalian cells (Dalakouras et al., 2006, Hogue and Nayak, 1994, Tanimoto et al., 2004, Yongkiettrakul et al., 2009). Unfortunately, due to low expression levels, large amounts of membrane-bound NA may be difficult to obtain. There is also a need to develop specialized purification schemes which could complicate further the production process. Alternatively, NA has been expressed in secreted form (sNA) to contain the enzymatic head domain fused to a secretion signal at its N-terminus (Yongkiettrakul et al., 2009). This soluble and catalytically active sNA was expressed successfully in Pichia pastoris. The protein displayed kinetic properties (K_m) and affinity for the inhibitor oseltamivir (K_i) comparable to those of NA on the virus surface. However, the inherent hyperglycosylation of protein expressed in yeast renders it a weak candidate for induction of proper anti-influenza immune responses (Martinet et al., 1997). On the other hand, sNA expressed in mammalian cells was reported to be able to elicit a protective immune response against lethal challenge with pathogenic influenza virus (Bosch et al., 2010). Mammalian cells are therefore considered a suitable platform for sNA expression due to glycosylation patterns that are physiologically relevant in the context of human infection. Recombinant protein expression in mammalian cells normally involves the generation of stable cell lines, requiring substantial time and resources. A transient gene expression (TGE) system serves as a good alternative due to its ability to express recombinant proteins rapidly and its capacity for large scale production (Durocher et al., 2002, Geisse, 2009, Majors et al., 2008). Nevertheless, the yield obtained from this system is still lower than that from stable cell lines, indicating a need for strategies to enhance protein expression in the TGE system (Backliwal et al., 2008).

One possible approach is the use of influenza virus nonstructural protein 1 (NS1), which plays multiple roles during viral replication, including regulating protein expression in infected cells (Hale et al., 2010). Several reports have shown that NS1 enhances expression of influenza viral proteins such as nucleoprotein (NP) and matrix (M) (de la Luna et al., 1995). The increase in protein expression due to NS1 is not limited to influenza virus genes, as expression of non-viral genes such as luciferase was also shown to increase in the presence of NS1 (Salvatore et al., 2002). Accordingly, NS1 may serve as an enhancer for sNA expression in the TGE system in mammalian cells.

This study describes a novel expression protocol for high-yield and rapid expression of sNA of avian influenza A (H5N1) virus in mammalian cells. The method combines the use of influenza virus NS1 to enhance H5N1 sNA expression with the TGE system in serum-free suspension cultures to obtain large amounts of H5N1 sNA. The obtained H5N1 sNA protein displayed comparable kinetic properties and glycosylation to those of native NA on the viral surface. Hence, this method could be used for rapid high-level expression of sNA of other influenza virus strains and, possibly, other recombinant proteins.