The Veterinary Journal 202 (2014) 612–617

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The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l

The amino-terminal region of the neuraminidase protein from avian H5N1 influenza virus is important for its biosynthetic transport to the host cell surface Guomin Qian a, Song Wang a, Xiaojuan Chi b, Hua Li a, Haitao Wei a, Xiaomei Zhu c, Yuhai Chen a, Ji-Long Chen a,b,* a

Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China c School of Life Sciences, Anhui University, Hefei 230601, China b

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Article history: Accepted 9 October 2014 Keywords: Avian influenza virus Neuraminidase H5N1 Protein intracellular transport Cdc42

A B S T R A C T

Influenza virus neuraminidase (NA) is a major viral envelope glycoprotein, which plays a critical role in viral infection. Although NA functional domains have been determined previously, the precise role of the amino acids located at the N-terminus of avian H5N1 NA for protein expression and intracellular transport to the host plasma membrane is not fully understood. In the present study, a series of N-terminal truncation or deletion mutants of H5N1 NA were generated and their expression and intracellular trafficking were investigated. Protein expression from mutants NAΔ20, NAΔ35, NAΔ40, NAΔ7-20 and NAΔ7-35 was undetectable by immunoblotting and by performing NA activity assays. Mutants NAΔ6, NAΔ11 and NAΔ15-20 showed a marked decreased in protein expression, whereas mutants NAΔ7-15 and NAΔ15 displayed a slight increase in protein expression, compared with that of the native NA protein. These data suggest that amino acid residues 16–20 are vital for NA protein expression, while amino acids 7–15 might suppress NA protein expression. In deletion mutants NAΔ7-15 and NAΔ15 there was an accumulation of NA protein at the juxta-nuclear region, with reduced expression of NA at the cell surface. Although active Cdc42 could promote transport of wild-type NA to the host cell surface, this member of the Rho family of GTPases failed to regulate transport of mutants NAΔ7-15 and NAΔ15. The results of the study reveal that amino acid residues 7–15 of H5N1 NA are critical for its biosynthetic transport to the host cell surface. © 2014 Elsevier Ltd. All rights reserved.

Introduction Influenza A viruses are a major cause of seasonal epidemics of respiratory disease and occasional global pandemics, associated with high levels of morbidity and mortality (Molinari et al., 2007; Gambotto et al., 2008; Medina and Garcia-Sastre, 2011). Influenza viruses consist of various subtypes that are classified based on their expression of two envelope glycoproteins, namely haemagglutinin (HA) and neuraminidase (NA) (Medina and Garcia-Sastre, 2011). To date, 18 HA (H1-H18) and 11 NA (N1-N11) influenza A virus subtypes have been identified, with most of these viruses circulating in avian species (Tong et al., 2013). Highly pathogenic avian influenza A virus (HPAIV) subtype H5N1 is endemic in some bird species, and has been transmitted from birds to humans following emergence in 1996 (Subbarao et al., 1998). Infection of humans with this particular avian influenza A virus causes

* Corresponding author. Tel.: +86 106 480 7300. E-mail address: [email protected] (J-L. Chen). http://dx.doi.org/10.1016/j.tvjl.2014.10.015 1090-0233/© 2014 Elsevier Ltd. All rights reserved.

severe respiratory disease and can result in mortality rates of up to 60% (Subbarao et al., 1998; Tran et al., 2004). Additionally, the H5N1 HPAIV is a significant threat to the poultry industry, causing high mortality on chicken farms (Shortridge et al., 1998). Although it is thought that mortality in humans associated with H5N1 infection arises as a consequence of an induced lymphopenia and cytokine storm (de Jong et al., 2006; Tran et al., 2004; Wei et al., 2014), the mechanisms that control infection in mammalian hosts remain elusive. Influenza virus infection of cells follows a process typically seen in enveloped viruses, involving receptor binding, internalisation, replication, virus assembly and budding (Ali et al., 2000; Nayak et al., 2004). Virus assembly and morphogenesis require transport of newly synthesised viral proteins, such as HA and NA, to the host cell surface. After assembly, the NA protein cleaves sialic acid moieties from sialyloligosaccharides and facilitates release of nascent virions (Air and Laver, 1989). Thus, NA, a type II glycoprotein, plays a critical role in infection. The NA protein consists of four distinct domains, specifically a cytoplasmic tail, a transmembrane domain (TMD), a stalk region,

G. Qian et al./The Veterinary Journal 202 (2014) 612–617

and a globular head (Barman et al., 2004; Air, 2012). The first six amino acids at the amino-terminus of NA are conserved among influenza A viruses and are thought to determine NA incorporation and virus morphogenesis (Mitnaul et al., 1996; Jin et al., 1997). Previous studies have shown that the TMD is involved in intracellular trafficking, oligomerisation, glycosylation, and lipid raft anchoring (Markoff et al., 1984; Brown et al., 1988; Barman and Nayak, 2000; da Silva et al., 2013). However, the precise role of the amino acids at the amino-terminus of the NA protein in its biosynthetic transport to the plasma membrane remains to be further elucidated. Increasing evidence suggests that the Rho family of GTPases is involved in intracellular transport of proteins (Hehnly et al., 2009; Mohammadi and Isberg, 2013). One of the best-characterised of these is Cdc42, which plays important roles in cell polarisation, membrane transport, endocytosis, Golgi apparatus positioning, and protein trafficking (Erickson and Cerione, 2001; Harris and Tepass, 2010). We have previously shown that Cdc42 participates in regulating influenza A virus replication, through its effects on intracellular trafficking of NA protein (Wang et al., 2012). However, it is still unclear which region of NA is critical for its intracellular transport, regulated by Cdc42. The aim of the present study was to investigate the role of the amino-terminal sequence of NA in its biosynthetic transport to the host cell plasma membrane. Materials and methods Plasmid construction Wild type NA of the A/Anhui/1/2005 (H5N1) virus strain (GenBank accession number: EU128239), and NA mutants, including NAΔ6, NAΔ11, NAΔ15, NAΔ20, NAΔ35, NAΔ40, NAΔ7-15, NAΔ7-20, NAΔ7-35 and NAΔ15-20, were subcloned into the pcDNA3.1(−) vector (Promega) with a HA tag at the carboxyl-terminus. Human Cdc42 was subcloned into the pEGFP-C1 vector and a mutant version, Cdc42 (Q61L), was generated using the QuikChange site-directed mutagenesis kit (Stratagene) to alter the coding sequence at codon 61 from glutamine to leucine. LacZ was subcloned into the CMV-5a-FLAG vector. A fusion construct of NA downstream of the human interleukin 2 signal peptide sequence (designated NA-IL2sp), designed to facilitate secretion of NA, was generated in the CMV-5a-FLAG vector and used as a control for examining NA activity in cell culture medium. Cell culture and transfection 293T cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/ mL penicillin and 100 U/mL streptomycin. Cell transfections were performed using VigoFect reagent (Vigorous Biotechnology) according to the manufacturer’s instructions, as previously described (Wang et al., 2012). Briefly, 293T cells were seeded into 6-well plates at 1 × 106 cells per well. Subsequently, 1.5 μg DNA and 2 μL VigoFect reagent were added to 200 μL DMEM (without serum or antibiotics) and mixed for 20 min. The mixture was then added to each well containing 1 mL DMEM and the cells cultured for a further 36 h. Antibodies and reagents The primary antibodies used in the study were anti-FLAG (Sigma–Aldrich), antiGFP (Sigma-Aldrich), anti-HA (Medical and Biological Laboratories) and antiGM130 (BD Bioscience). Dylight 488-conjugated Affinipure donkey anti-rabbit and Dylight 594-conjugated Affinipure donkey anti-mouse antibodies (Jackson ImmunoResearch Laboratories) were used as secondary antibodies. All other antibodies were as described previously (Guo et al., 2010). Analysis of NA activity An NA enzymatic activity assay was conducted as previously described (Wang et al., 2012). Briefly, 293T cells expressing wild-type NA or its mutants were resuspended in assay buffer (15 mM MOPS, 145 mM NaCl, 2.7 mM KCl and 4.0 mM CaCl2, pH 7.4) containing 2% FBS. Alternatively, cell-free supernatants were collected from transfected cells. Samples were analysed using a Neuraminidase Assay Kit (Beyotime Institute of Biotechnology), whereby 5 × 104 cells resuspended in 10 μL assay buffer were mixed with 70 μL detection buffer, 10 μL NA fluorogenic substrate and 10 μL water. Cleavage of the fluorogenic substrate by NA produces fluorescence with an emission wavelength of 440 nm and an excitation wavelength of 360 nm, measured using a multifunctional microplate reader (SpectraMax M5, Molecular Devices).

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NA activity is shown as the intensity of fluorescence above the background values for non-transfected cells. Immunofluorescence and immunoblotting experiments Immunofluorescence assays were performed as previously described (Chen et al., 2005; Guo et al., 2010). Briefly, Hela cells transfected with plasmid DNA containing wild-type or mutant NA for 36 h were fixed with 4% paraformaldehyde, incubated with primary antibodies for 2 h, followed by 1 h incubation with secondary antibodies. Images were obtained using a confocal microscope (model LSCMFV-500) and a 60× objective (Olympus Optical) with a numerical aperture of 1.40. Western blotting was carried out as described previously (Chen et al., 2005; Guo et al., 2010). Briefly, 1 × 106 293T cells transfected with plasmid DNA encoding wildtype NA or its mutants were lysed with 40 μL cell lysis buffer. Cell lysates (10 μL) were separated on SDS-polyacrylamide gel, transferred onto PVDF membranes and probed with the indicated antibodies. NA immunoreactivity was quantified by densitometry and normalised against β-actin expression. Analysis of NA mRNA expression Total RNA was isolated from 293T cells, transfected with plasmid DNA encoding wild-type NA or its mutants using Trizol reagent (Invitrogen). Reverse transcription to cDNA was performed using M-MLV reverse transcriptase (Promega) and oligo (dT) primers. The NA mRNA expression was detected by PCR, with expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA used as a control. The following primers were used: NA forward, (5′-CCAAGCTGAACCAATCAGGAA-3′), NA reverse, (5′-CCATTGGAGTGCTTGTCATTC-3′), GAPDH forward, (5′-AGAAGGCTG GGGCTCATTTG-3′) and GAPDH reverse, (5′-AGGGGCCATCCACAGTCTTC-3′). One hundred nanograms of cDNA was mixed with 0.5 U rTaq polymerase (Takara), 1.5 μL buffer (500 mM KCl, 15 mM MgCl2 and 100 mM Tris–HCl, pH 8.3), 1.2 μL dNTP mixture (2.5 mM each), 1 μL primers (10 μM each) and molecular grade water to a final volume of 15 μL. PCR was then performed for 30 cycles of 95 °C for 30 s, 53 °C for 30 s and 72 °C for 30 s using a MyCycler (Bio-Rad) thermocycler. Statistical analysis The mean ± SE of three or more independent experiments is reported. Statistical significance was determined using Student’s t test, with a P value