The first reported human infected with the highly pathogenic avian influenza (HPAI) H5N1 virus was recorded in Hong Kong, 1997 [1
]. Subsequently, the spread of HPAI H5N1 viruses in poultry and sporadic human infections are perceived as a potential pandemic threat [2
]. At present, there are several clades and subclades being identified based on their genetic and antigenic variation of the viral haemagglutinin (HA5) and neuraminidase (NA1) genes. Phylogenetic analysis indicates that some of the 10 first-order clades (0–9) have ceased to circulate since 2008 or even earlier (clades 0, 3, 4, 5, 6, 8, 9), as have some second- and third-order groups of clades 2. Meanwhile, clades 1, 2.1.3, 2.2, 2.2.1, 2.3.2, 2.3.4, and 7 have continued to evolve [4
]. Among them, clade 2.3.2 is widely distributed in Asian countries. Most H5N1 human infection results from direct contact with infected poultry [5
]. Between 2003 and October 2020, there have been 861 human cases and 455 deaths reported by the World Health Organization (WHO) [6
Influenza A viruses belong to the Orthomyxoviridae family. Their genome consists of eight negative stranded RNA segments, which encode for more than 10 major proteins [7
]. The influenza A virion is studded with glycoprotein spikes of hemagglutinin (HA) and neuraminidase (NA), in a ratio of approximately four to one, projecting from a host cell-derived lipid membrane [8
]. In addition, a smaller number of matrix (M2) ion channels traverse the lipid envelope. The envelope and its three integral membrane proteins HA, NA, and M2 overlay a matrix of M1 protein, which encloses the virion core [9
]. The attachment of influenza A virus to sialic acids (SAs) on the cell surface is a critical first step in the initiation of infection. SAs are an essential factor for the tropism of the influenza virus since their type of linkage to the galactose residue determines whether they are recognized by the specific viruses. SAs are widely expressed in most tissues and organs. Avian influenza A viruses (AIVs) have a preference in recognizing sialo-sugar chains terminating in sialic acid-α2,3-galactose (SAα2,3Gal), whereas human influenza A viruses prefer SAα2,6Gal. Regarding the pathogenesis of H5N1, regardless of the exposure route, the majority of cases develop viral pneumonia [3
]. During the initial phase of human H5N1 infection, the viruses are rarely isolated from the upper respiratory tract, most likely due to high abundance of SAα2,3Gal on the cells of the lower respiratory tract in humans as opposed to the upper respiratory tract [10
]. The cases of H5N1 infection have recorded a high mortality rate (~60%) [3
]. The disease severity of H5N1 in human may be induced by several factors, such as virulence, strain type, cytokine storm, and immune dysregulation [12
]. In addition to lung dysfunction, systemic infection and multiple organ failure are often observed in the H5N1 fatal cases [13
]. However, the detailed mechanism regarding severe H5N1 infection remains unclear.
In recent decades, lectins have proven to play critical roles in regulation of microorganisms’ infection [16
]. DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) is a calcium-dependent lectin, with a wide range of biological functions. High-level expression of DC-SIGN has been demonstrated on immature DC and macrophage subpopulations abundant in the dermis of the skin, at mucosal surfaces, and in lymph nodes and peripheral tissues [21
]. In addition to regulate DC migration and T-lymphocyte activation via interaction with ICAM-3, expression of DC-SIGN has been reported to mediate and enhance many viral infections, such as HIV-1 [22
], HCV [23
], Ebola virus [24
], dengue virus [25
], Coronavirus [26
], and influenza virus [27
]. DC-SIGN has been reported to interact with specific carbohydrate structures on pathogens to internalize pathogens via clathrin-mediated endocytosis for degradation in lysosomal compartments to enhance antigen processing and presentation [21
]. Regarding HIV-1, DC-SIGN binds to the highly glycosylated HIV envelope (Env) gp120 in a CD4-independent fashion and can efficiently transfer the virus to CD4+ permissive T cells, thereby facilitating viral infection in tran
Influenza HA is a highly glycosylated protein [32
]. Cellular lectin receptors may recognize the glycans on HA of influenza A virus, allowing for the binding of the virus to the cells and permitting internalization. Thus, the extent of glycosylation of HA is likely important for the recognition of the virus by cellular lectins. Glycosylation is achieved by post translational modification of Asparagine residues of the NXS/T motif (X can be any amino acid except Proline) [33
]. Numbers, types, and the positions of glycans vary for each virus, which might affect recognition of influenza viruses by the lectin receptors such as DC-SIGN. We were the first to reported that DC-SIGN acts as an attachment molecule which facilitates H5N1 infection, in addition to assist in H5N1 dissemination to the other susceptible cells [29
]. Following our findings, several studies indicate that DC-SIGN plays an important role in H5N1 infection and H5N1 pathogenesis [27
]. Furthermore, Londrigan et al. even reported DC-SIGN as an alternative receptor for influenza H1N1 entry [28
] and Hillaire et al. reported that in the absence of sialic acids, human influenza A viruses can replicate in DC-SIGN expressing cells, indicating that efficiency of DC-SIGN mediated infection is dependent on the extent of glycosylation of the viral hemagglutinin [27
Currently, the detail mechanism of severe pathogenesis caused by H5N1 AIVs infection remains unclear. We proposed that the interaction between DC-SIGN and HA may play an important role in regulation of H5N1 AIVs infection and transmission. The essential N-linked glycosylation sites on HA are the main factors to modulate this interaction. In this study, we aimed to identify important DC-SIGN interaction with the N-linked glycosylation sites on HA of H5N1 AIVs.
The binding of influenza A viruses to cells are not restricted to the recognition of sialic acids by the receptor binding site (RBS) of HA. It has been reported that influenza A viruses can bind to lectin receptors, suggesting that lectin-HA interaction might be involved in virus attachment and subsequent viral entry [37
]. We previously addressed the role of DC-SIGN in H5N1 AIVs infection and demonstrated that DC-SIGN can serve as an attachment molecule to facilitate H5N1 infection [29
]. In this study, we extended the previous findings to further identify the important DC-SIGN interaction with the N-liked glycosylation sites in HA of H5N1 AIVs. Our results indicate that N27 and N39 are two essential N-linked glycosylation sites in HA protein which are involved in DC-SIGN interaction in H5N1 AIVs. Mutations on these two N-glycosylation sites significantly reduced HA binding to DC-SIGN, and ameliorated DC-SIGN positive regulatory effects on H5N1 infection in cis
and in trans
. This is the first study to address the optimal N-glycosylation sites in HA of H5N1 AIVs.
DC-SIGN is a transmembrane C-type lectin receptor with a long extracellular neck region and a carbohydrate recognition domain (CRD) [21
]. DC-SIGN is known to have high affinity to N-linked high-mannose oligosaccharides and branched fucosylated structures [39
]. The N-linked glycans are found commonly on viruses, bacteria, and fungi [21
]. Our results suggest that almost all human H5N1 isolates from clade 0 to clade 7, during 2004–2020, contain the same pattern and numbers of N-linked glycosylation sites on the HA protein (Figure 1
and Figure 2
). Previous studies have shown that N-linked glycosylation on HA of human influenza A viruses could assist to hide viral antigen to reduce immune detection and attack [21
]. Under circulation and evolution, the pattern and numbers of N-glycosylation sites have been altered and modified in human influenza A viruses (such as H1N1 and H3N2) as reported in previous studies [7
]. However, these changes were not found in the human H5N1 isolates used in this study. Accordingly, we suggest that H5N1 has not evolved in humans for a long time and this N-glycosylation pattern found in H5N1 might assist the viruses to escape immune attack, and additionally, to interact with lectins such as DC-SIGN to enhance viral infection.
Currently, H5N1 AIVs has reported high mortality in human infection and is listed as a bio-safety level (BSL)-3 pathogen. Owing to lack of such facility, the pesudotyped virus system (belonged to BSL-2 pathogen) was used in this study. The H5N1-PVs were generated using a vector expressing lentivirus core structure combined with vectors expressing HA5 and NA1 proteins (Figure 3
). The hemagglutination and infectivity of H5N1 pesudotyped viruses were validated and immuno-TEM was utilized to further prove the similarity of the virological function with avian H5N1 viruses (Figure 3
). A similar strategy has also been used in several previous publications [29
], suggesting that the lentivirus particles with the H5N1 envelope is a suitable tool allowing for handling in a common virological laboratory.
Firstly, we screened for the important N-glycosylation sites expressed on HA of H5N1 virus by rDC-SIGN protein coated ELISA, to interact with H5N1-PVs bearing different mutagenetic N-liked glycosylation sites on HA. A similar strategy was used in a previous study by Hong et al. [44
], who used rDC-SIGN proteins to screen HIV-1 viruses carrying different N-glycosylation mutations on HIV-1 gp120 envelope. In this study, we found that N27, N39, and N181 on HA could be critical in interacting with rDC-SIGN proteins using ELISA assay. These three N-glycosylation sites were located on the side and top region of HA1 structure. Further, we used monocyte derived iDCs and mDCs to confirm these findings and found that only N27Q and N39Q mutations significantly reduced DC-SIGN mediated H5N1 virus infection. However, this amelioration effect was not observed in H5N1 virus carrying N181Q mutation. We suggest that the interaction between HA and DC-SIGN might be influenced by the quality and purity of rDC-SIGN proteins in the ELISA. In addition, the native form of DC-SIGN is a tetrameric transmembrane protein. Accordingly, we propose that using DC-SIGN expressing cells are better and mimic the real-life situation compared to using artificial rDC-SIGN proteins. Nevertheless, several studies still suggest that rDC-SIGN is a good candidate for fast screening of important DC-SIGN interactive N-linked glycosylation sites [26
Currently, the detailed interaction and binding mechanism between DC-SIGN and distribution or distance of N-linked glycan sites remains controversial. DC-SIGN is known to bind to two classes of carbohydrate structures: N-linked high mannose oligosaccharides (such as Man9GlcNAc2) and branched, fucosylated oligosaccharides [45
]. High-mannose glycans are abundantly detected on many types of enveloped viruses [47
Menon et al., reported that crystal structures of DC-SIGN CRD complex with oligosaccharide ligands revealed that extensive ligand-binding site in DC-SIGN results in higher-affinity of one-to-one interactions with specific glycans (e.g., DC-SIGN CRD forms a one-to-one complex with a high-mannose oligosaccharide), although engagement of such ligands is more geometrically constrained [48
]. The oligosaccharide is known to only be accommodated in a single orientation, thus this specificity places spatial constraints for DC-SIGN interaction with high-mannose glycans on pathogen surfaces [49
]. Other studies also indicate that DC-SING with flexibility to bind to the ligands, allows multiple CRDs in a tetramer to interact with more complex but more sparsely spaced glycan ligands on a membrane [48
]. In addition, Medina et al. noted that the N-glycosylation of the influenza hemagglutinin plays an important role in the life cycle of influenza virus and plays a key role on its antigenic fitness. Indeed, oligosaccharides attached to the globular head of HA have been shown to modulate virus antigenic properties and its receptor binding [50
]. Our results demonstrated that most single mutations on N-linked glycosylation sites on HA5 did not influence the binding between DC-SIGN and HA (H5), whereas only three mutations (N27Q, N39Q, and N181Q) reduced this interaction; suggesting that the DC-SIGN CRD may interact minimally with a N-glycosylation with high-mannose oligosaccharides expression on HA protein. When it came to N181, which was located on the top side of HA structure, this interaction might only be displayed with a high dose of recombinant monomeric DC-SIGN protein instead of tetrameric native structure. We suggested that this interaction might be non-specific or weak which led to no significant interaction with tetrameric form of DC-SIGN expressing cells. The details for this phenomenon require further investigation.
We also noted that combination of two N-glycosylation mutations on HA additively ameliorated DC-SIGN binding to HA5, and reduced DC-SIGN-mediated promoting effect on H5N1 cis
infection. Previously, Zhang et al. reported that soluble DC-SIGN bound to dengue virus, suggesting that the CRD monomer can also bind to two N-glycosylation sites spaced 18 Å apart on adjacent envelope dimers on the virion surface using cryo-electron microscopy analysis, proposing that the CRD has flexibility in the interaction with two N-glycosylation sites [51
]. The two carbohydrate moieties bind to a single CRD monomer, which might be a result of the elongated oligosaccharide binding valley present in the CRD of DC-SIGN [42
]. Another study noted that the distance between N-glycosylation sites was approximately 19 to 23 Å, thus fulfilling the minimal spatial requirement for at least two N-glycosylation sites for binding to a single DC-SIGN CRD [44
]. Our results estimated that the distance between N27 and N39 was around 18.6 Å (Figure 8
). This distance is similar to results found in dengue virus. Accordingly, we suggest that the distance (18.6 Å) between two identified N-linked glycosylation sites in this study still fit the minimal requirement regarding DC-SIGN binding.
There were some limitations to this study. We simply expressed the recombinant extracellular domain of DC-SIGN for the screening of the N-linked glycosylation sites. The purity of rDC-SIGN proteins may influence their interaction with interactive proteins, especially those weak interactive proteins. Most of the results in this study used the H5N1 PVs or H5N1-RG. Although these viruses were reported as representative candidates for studying the function of HA or NA, the real H5N1 AIVs are still necessary to validate and confirm the findings in this study. Because it might be worth considering the glycomics of the different cell lines and how glycosylation of the H5N1 PV particle released from HEK293T cells may not be the same as glycosylation of the HA of the virus grown in other cellular substrates or in natural infection, and so the DC-SIGN recognition may be related to the system used to generate the virus. Further, the fine-tune mechanism regarding how DC-SIGN regulates these H5N1 mutants containing different N-glycosylation mutations was not performed. The detail mechanism including DC-SIGN mediated endocytosis, as well as the downstream signaling pathway, are worthy of further investigating.
In this study, we generated recombinant DC-SIGN proteins which were used to establish ELISA based screening assay to map essential N-linked glycosylation sites on HA protein of H5N1 viruses. Our data demonstrated that N27 and N39 are two essential N-glycosylation sites, which are majorly involved in the binding to the DC-SIGN. Mutation on these two N-glycosylation sites on HA significantly ameliorated DC-SIGN mediated H5N1 infection in cis and in trans. Further investigation is demanded to understand the DC-SIGN regulatory mechanism on H5N1 AIVs and even other subtypes of influenza A virus infection.
4. Materials and Methods
4.1. Ethics Statement
Written informed consent was elicited from all study participants. Approval was applied for and received from the Institutional Ethics Committee of the Kaohsiung Medical University, Taiwan. All procedures were conducted according to committee regulations.
HEK293T (human kidney), Madin-Darby canine kidney (MDCK), Raji B, Raji-DC-SIGN (a stable B-THP-1 clone expressing DC-SIGN molecules), THP-1 (monocyte), and THP-1-DC-SIGN (a stable THP-1 clone expressing DC-SIGN molecules) were used in this study. HEK293T and MDCK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Rockville, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone™ Characterized Fetal Bovine Serum, U.S.), penicillin (100 U/mL), streptomycin (100 μg/mL), non-essential amino acids (0.1 mM), and L-glutamine (2 mM) (GIBCO-BRL). Raji, Raji-DC-SIGN, THP-1, and THP-1-DC-SIGN cells were cultured in RPMI 1640 medium (GIBCO-BRL) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). For the B-THP-1/DC-SIGN cell line, 50 μg/mL of neomycin (Sigma-Aldrich) was added to the medium.
4.3. H5N1 Pseudotype and Reverse-Genetic Virus Preparation
The full-length of HA and NA from A/Vietnam 1203/04 were amplified using RT-PCR with the primers containing the universal conserved non-coding regions of influenza A virus (AGCAAAAG CAGG or AGTAGAAACAAGG). The details are described elsewhere [53
]. The primers used in the PCR reaction contained segment specific sequences and Bsm
BI or Bsa
I restriction site sequences at their end. After digestion of the PCR products with Bsm
BI or Bsa
I, the fragments of HA5 and NA1 were cloned into the vector, pHW2000. The H5N1 pseudo-type viruses (PVs) were generated using co-transfection of pNL-Luc-E-R- vector with pHW1203-HA and pHW1203-NA vectors into HEK293T cells. Cell supernatant containing H5N1-PVs were collected 48 h post-transfection and purified through a 0.45 μm filter. Supernatant was concentrated by ultracentrifugation at 25,000 rpm for 2.5 h to obtain high concentration of H5N1 PV particles. Regarding H5N1 reverse-genetic virus, the detailed protocol is described elsewhere [53
]. Briefly, the two plasmids encoded HA and NA of A/Vietnam/1203/04 strain and the other six plasmids encoded structure and non-structure proteins of A/Puerto Rico/8/34 stain were co-transfected into HEK293T and incubated at 37 °C for 48 h. The viral supernatants were collected and subjected to propagation in Vero or MDCK. The H5N1-RG was used as vaccine candidate and could be handled in biosafety level 2 facility (BSL-2) [54
4.4. N-Linked Glycosylation Site Prediction
Predictions of N-linked glycosylation on H5N1 hemagglutinin protein were performed using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/
). The full length of hemagglutinin amino acid sequence from the influenza A H5N1 (A/Vietnam/1203/04 strain; accession no. ABW90135) and other human H5N1 isolates from 2004–2020 searched and downloaded from NBCI Influenza Resource (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/genomes/FLU/Database/nph-select.cgi?go=database
). There was a total of 274 human H5N1 isolates obtained and most H5N1 isolates were found during 2003–2015. After collapsing identical sequence, all the HA sequences were uploaded to the website and total N-linked glycosylation sites were predicted following the Asn-X-Ser/Thr rule. The 3D hemagglutinin structure file was downloaded from the Protein Data Bank (file name: 2FK0). N-linked glycosylation sites were labeled on the structure shown via the DS ViewerPro 5.0 program.
4.5. Mutagenesis of N-Linked Glycosylation Site of HA (H5) Protein
To mutate the N-linked glycosylation sites from Asn (N) to Gln (Q), the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used. The detailed protocols were published elsewhere [55
]. Forward and reverse primers were designed for the predicted N–linked glycosylation sites. For this study, overlapping PCR was done on pHW1203-HA, following by treatment with Dpn
I restriction endonuclease digestion for 2 h at 37 °C. The pHW1203-HA with different N-linked glycosylation mutations including N26Q, N27Q, N39Q, N170Q, N181Q, N209Q, N302Q, N500Q, N599Q and other combination of mutations were generated. All of the plasmids were confirmed by sequencing using a dye terminator cycle sequencing core kit on a DNA sequencer (Applied Biosystems, Foster City, CA, USA).
4.6. Immuno-Electron Microscopy (Immuno-EM)
The detailed protocol of immuno-EM was described in previous studies [56
]. Briefly, pNL-Luc-E-R-, pHW1203-HA, and pHW1203-NA vectors were transfected to HEK293T (2:1:1 ratio) and incubated at 37 °C for 48 h. The viral supernatants and cell pellets were collected for immuno-EM staining. Supernatants were filtered (0.45-mm mesh), placed on 20% sucrose, and subsequently ultracentrifuged at 100,000× g
for 2 h; cells were resuspended in 50 μL PBS. Viral droplets were placed on grids and fixed with 0.5% glutaraldehyde and 4% paraformaldehyde for 10 min. Grids were washed three times with PBS and blocked with 1% fish gelatin and held at room temperature for 30 min. Immunolabeling was performed using primary mouse anti-HA (H5) monoclonal antibodies and incubated overnight at 4 °C. After three PBS washes, grids were incubated with secondary antibodies labeled with 10-nm gold particles (goat anti-mouse IgG polyclonal Ab) and held for 1 h at 37 °C. After three additional PBS washes, grids were stained with 4% uranyl acetate and lead citrate. Electron micrographs were obtained using a Hitachi H-7000 Transmission Electron Microscope.
The pcDNA3/hIgG1.Fc(mut)-DC-SIGN.ECD vector was kindly offered by Dr. Jason C. Huang at National Yang Ming University. This plasmid expressing recombinant DC-SIGN extracellular domain was produced in 200 mL suspension cultures of the FreeStyle™ 293-F cells in FreeStyle 293 Expression Medium (Gibco). The detailed protocol is described elsewhere [58
]. Equal amounts of purified recombinant DC-SIGN proteins (1 μg/well) were coated on 96 well plate and then blocked by 5% bovine serum albumin [BSA]. Next, the H5N1-PVs wild-type or mutants, with different N-linked glycosylation mutations, were incubated with recombinant DC-SIGN protein coated ELISA to test their binding abilities to DC-SIGN for 2 h at 37 °C. After three washing with PBST, the anti-p24 monoclonal antibody (1:1000) was used to detect the binding H5N1-PVs.
4.8. Immunofluorescent Staining
For confirmation of expression of DC-SIGN, the pcDNA3/hIgG1.Fc(mut)-DC-SIGN.ECD vectors were transfected to 293-F cells and incubated at 37 °C for 48 h. The cells were subjected to immunostaining with mouse anti-DC-SIGN monoclonal antibodies (R&D, Cat. No. MAB161) (1:1000) and incubated at room temperature for 1 h. After three washes with PBS, the cells were strained with goat anti-mouse-IgG conjugated Alexa555 (Abcam, Cat. No. ab150118) (1:1000). After repeating the PBS wash, the stained cells were observed using a Carl Zeiss LSM 700 Laser Scanning Confocal Microscope. For observation of the H5N1-PVs binding to DC-SIGN expressing on the cells, Raji and Raji-DC-SIGN cells were pre-treated with sialidase (0.25 U/mL) for 1–3 h and these cells were incubated with H5N1-PVs at 4 °C for 2 h. After washing with PBS, these cells were fixed with 4% paraformaldehyde fixation and blocked with 1% BSA. The cells were then stained with rabbit anti-HA5 polyclonal antibodies (in house preparation) (1:1000) and mouse anti-DC-SIGN monoclonal antibodies (R&D, Cat. No. MAB161) (1:1000), followed by the addition of fluorescently labeled antibodies against the primary antibodies. After mounting, stained cells were observed using a Carl Zeiss LSM 700 Laser Scanning Confocal Microscope.
4.9. Cis Infectivity Assay
The equal amounts of H5N1 pseudotyped virus particles (quantification with a Coulter HIV-1 p24 antigen assay (Beckman Coulter)) were collected and incubated with Raji, Raji-DC-SIGN, THP-1, THP-1-DC-SIGN cells, as well as immature DC (iDC) and mature DC (mDC). Luciferase activity was measured in cell lysate after 48 h of incubation at 37 °C. For a DC-SIGN-enhanced infectivity assay, a 5 × 105
susceptible cells mentioned above were seeded into 48-well plates prior to incubation with H5N1 pseudotyped or H5N1-RG virus particles at 37 °C for 2 h. Alternatively, some of these cells were pretreated with anti-DC-SIGN monoclonal antibodies (10 μg/mL-1; R&D System, catalog no. MAB161). After incubation, virus-bound cells were washed 3 times with PBS and harvested. Quantities of pseudotyped H5N1 or RG strain particles were determined by real-time reverse transcription (RT)-PCR (for detecting H5N1 gene expression) [59
] and luciferase activity (for the pseudotyped virus particles) [26
4.10. Trans Infectivity Assay
DC-SIGN-mediated virus transfer efficiency was assessed by capture assay as described previously [26
]. Briefly, the 5 × 105
captured cells (Raji, Raji-DC-SIGN, THP-1, THP-1-DC-SIGN, iDC and mDC) were incubated with H5N1-PVs (50 ng p24 equivalent) or 100 uL of H5N1-RG viruses (105
viral RNA copies/mL) at 4 °C for 2 h for virus binding, then washed with ice-cold PBS three times before being added to the target MDCK cells. Target cells were harvested after co-culturing for 18–24 h or 24–48 h. H5N1 viral RNA quantification was performed using qRT-PCR. Quantification of the H5N1-PVs titers was achieved using a luciferase reporter assay kit (Promega, Madison, WI, USA) and a luminometer (PerkinElmer, Waltham, MA, USA). For controlling the budding virions from cis
infected captured cells and then caused additional infection to the target cells. The transwell system (Corning Costar, Amsterdam, The Netherlands), which separated virions captured cells (upper channel) and target cell (lower channel) was used to monitor this phenomenon. The relative infectivity of the virus transmitted target cells was determined by the viral titers measured in co-cultured target cells, normalized with the viral titers measured in the target cells from the transwell system.
Different cells or cell lines used in this study were infected with H5N1-RG viruses. The infected cell lysates were collected and subjected to RNA extraction using TOOLS EsayPrep total RNA kit (Cat. No. DPT-BD19, BIOTOOLS Co., Ltd., Taipei, Taiwan). After RNA extraction, reverse transcription was performed on the isolated RNA using the ToolsQuant II Fast RT kit (Cat. No. KRT-BA06, BIOTOOLS Co., Ltd., Taipei, Taiwan). The cDNA was further subjected to quantitative real-time PCR using the TOOLS SuperFast SYBR qPCR reagent (with ROX dye) (Cat.No.FPT-BB07, BIOTOOLS Co., Ltd., Taipei, Taiwan) with ABI 7000 real-time PCR machine. The primers for detection of specific HA gene of H5N1 virus were listed in previous studies [29
]. H5N1 viral quantities were calculated by interpolation from a standard curve generated by the parallel running of serial dilutions of known quantities of the H5 segments of cloned plasmids.
4.12. Generation of Monocyte-Derived Dendritic Cells (MDDCs)
For preparation of monocyte-derived dendritic cells, the protocol used in this study was published elsewhere [26
]. Briefly, the peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Umea, Sweden) reagent. Further, the monocytes were extracted from PBMCs with anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) via standard density gradient centrifugation. Isolated human monocytes were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum, 800 U/mL human granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA), and 500 U/mL human interleukin-4 (R&D) for 6 days to trigger differentiation into immature DCs (iDCs). iDCs were confirmed with CD1a, CD40, CD54, HLA-DR, CD80, CD83, CD86, and DC-LAMP cell markers by flow cytometry and morphological characteristics. To induce the transformation of iDCs into mature DCs (mDCs), 0.1 ug/mL lipopolysaccharide (LPS) was added to the RPMI 1640 medium for 1.5 days. mDCs were also confirmed using the above-mentioned cell markers and morphological characteristics. The DC-SIGN expression levels between iDCs and mDCs were measured by flowcytometry.
4.13. Apoptosis Assays
H5N1-RG induced cell apoptosis assays were performed using Annexin V and propidium iodide staining [60
]. The detail protocol was described elsewhere. Briefly, iDCs or mDCs were infected with H5N1-RG which carried different N-linked glycosylation mutations. The infected cells were stained with FITC-conjugated Annexin V (Calbiochem) according to manufacturer’s recommendations. Annexin V-FITC was diluted in the manufacturer’s Hepes-buffer (containing 2.5 mM CaCl2), added to the cultures, and incubated for 15 min at room temperature. Further, the cells were then fixed with 1% paraformaldehyde, permeabilized with 0.1% Triton, and then incubated with 2.5 ug/mL propidium iodide (PI) in PBS containing 1.2 uL/mL DNAse-free RNAse. The cells stained by Annexin V and PI were subjected to FACS analysis and observed under fluorescence microscope (Zeiss).
4.14. Hemagglutination Assay (HA)
The hemagglutination activity of H5N1 pseudtotyped viruses (PVs) carrying different N-glycosylation mutations was performed using 0.5% turkey RBCs as described previously [61
]. Briefly, the erythrocytes from turkey were suspended in phosphate-buffered saline (PBS), pH 7.4. In a U-bottomed 96-well plate, 50 μL of 10-fold dilution of purified H5N1-PVs and 50 μL of turkey RBCs were added and gently mixed. The reaction mixture was incubated at room temperature for 45 min. The virus caused hemagglutination was observed.
4.15. Statistical Analyses
All experiments were performed at least three times each. Statistical analyses were preformed using GraphPad Prism software. Statistical significance (p < 0.05) was calculated using unpaired Student’s t-tests.