- Open Access
Analysis of hemagglutinin-mediated entry tropism of H5N1 avian influenza
- Ying Guo†1, 2,
- Emily Rumschlag-Booms†1,
- Jizhen Wang†1,
- Haixia Xiao3,
- Jia Yu4,
- Jianwei Wang5,
- Li Guo6,
- George F Gao3,
- Youjia Cao4,
- Michael Caffrey7 and
- Lijun Rong1Email author
© Guo et al; licensee BioMed Central Ltd. 2009
- Received: 12 February 2009
- Accepted: 02 April 2009
- Published: 02 April 2009
Avian influenza virus H5N1 is a major concern as a potential global pandemic. It is thought that multiple key events must take place before efficient human-to-human transmission of the virus occurs. The first step in overcoming host restriction is viral entry which is mediated by HA, responsible for both viral attachment and viral/host membrane fusion. HA binds to glycans-containing receptors with terminal sialic acid (SA). It has been shown that avian influenza viruses preferentially bind to α2,3-linked SAs, while human influenza A viruses exhibit a preference for α2,6-linked SAs. Thus it is believed the precise linkage of SAs on the target cells dictate host tropism of the viruses.
We demonstrate that H5N1 HA/HIV pseudovirus can efficiently transduce several human cell lines including human lung cells. Interestingly, using a lectin binding assay we show that the presence of both α2,6-linked and α2,3-linked SAs on the target cells does not always correlate with efficient transduction. Further, HA substitutions of the residues implicated in switching SA-binding between avian and human species did not drastically affect HA-mediated transduction of the target cells or target cell binding.
Our results suggest that a host factor(s), which is yet to be identified, is required for H5N1 entry in the host cells.
- Human Immunodeficiency Virus
- Influenza Virus
- Sialic Acid
- H5N1 Virus
- Avian Influenza Virus
H5N1 is an avian influenza virus which originally circulated in aquatic birds without causing major disease. However, a rapidly spreading variant(s) of H5N1 is highly pathogenic to avian species, causing a major economic loss due to culling of millions of potentially infected birds [1–5]. Alarmingly, this virus has crossed the species barrier to cause numerous human (and other animal) fatalities in certain regions of Asia, Europe and Africa[6, 7]. The unprecedented spread and the high mortality rate of this virus have raised a major concern for a potential global pandemic. The lack of effective vaccines for humans and the emergence of oseltamivir-resistant H5N1 strains[8, 9] underscore the urgent need in developing novel prophylactic and therapeutic treatments against this virus.
Influenza virus is an enveloped, negative-stranded, and segmented RNA virus. Two viral glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the viral surface, determine antigenic subtypes. Although the role of NA in the influenza life cycle is not clear, one of its functions is to release the progeny viral particles from the cell surface during budding. In contrast, the roles of the prototypic HA in viral entry have been well characterized by molecular, biochemical, biophysical, and structural techniques. HA is synthesized as a precursor, HA0, that forms trimers in the endoplasmic reticulum (ER). This precursor is cleaved into two subunits, HA1 and HA2, which are linked by a disulfide bond. Many HAs contain a consensus sequence R-X-R/K-R as the cleavage site recognized by host furin-like proteases. The presence of this polybasic motif in HA has been shown to correlate with the high pathogenicity of influenza viruses[3, 10, 12–14].
The two subunits of HA perform distinct functions in viral entry. HA2 mediates membrane fusion and viral entry, while HA1 is involved in binding to the sialic acid (SA) receptors on the target cells. Comparison of the HA1 and HA2 sequences among influenza virus subtypes reveals that the HA2 sequence is well conserved, suggesting a highly conserved membrane fusion mechanism. However, the HA1 sequence is much more divergent, suggesting differences in affinity of receptor-binding and in antigenicity. Although HA1 binds to SA with a low affinity, it is believed that interaction of multiple HA molecules on the viral surface with the SA-containing glycoproteins or glycolipids on the cell surface increases the avidity of influenza virus to the target cells, and this interaction facilitates viral infection through endocytosis in a pH-dependent manner.
Although bird flu H5N1 is a highly contagious pathogen in avian species, its transmission to humans, or more rarely human-to-human transmission, has been very limited thus far. A more transmissible and sustained variant(s) of H5N1 in human populations may arise through accumulating mutations in multiple viral proteins of H5N1 and/or genomic reassortment between H5N1 and other influenza viruses. An outbreak of a highly pathogenic H5N1 influenza virus in migratory birds of several species in Qinghai Lake, China was reported recently[16, 17]. It was feared then and realized now that the H5N1-infected migratory birds could carry and transmit the virus to avian and non-avian animals including humans in densely populated areas on different continents. In this study, we characterized several aspects of the host cell tropism of H5N1, and our results implicate an unidentified host factor in H5N1 entry.
H5N1 HA can mediate HIV pseudoviral infection
Neuraminidase treatment of the producer cells enhancesHA-mediated viral infectivity
To improve and optimize the HA-mediated transduction efficiency, a commercially available neuraminidase, which catalyzes hydrolysis of α2,3, α2,6, and α2,8 linked N-acetyl-neuraminic acid residues from glycoproteins and oligosaccharides, was used to treat the transfected 293T producer cells twice at 26 and 46 hours post-transfection with ramping concentrations of 0, 5, 10, 20, 50, 100, and 200 units/ml. The collected viral supernatants were used to challenge susceptible target cells, 293T and A549 cells (a human lung cell line, see below), and the luciferase activities of the target cells were determined at 48 hours post-infection (Fig. 1B). The target cells challenged with the viral supernatants collected from the neuraminidase-treated producer cells, even at the lowest concentration used (5 units/ml), gave luciferase activities at least 10-fold higher than the same target cells challenged with the supernatants from the non-treated producer cells (5.21 vs 4 logs in 293T cells, and 4.49 versus 3.43 logs in A549 cells, respectively). The viral supernatants collected from the producer cells treated with higher concentrations of neuraminidase (10–200 units/ml) could further boost the luciferase signals in the target cells, up to approximately another 10-fold with the highest concentration used (5.21 vs 6.04 logs in 293T cells, and 4.49 vs 5.93 logs in A549 cells, respectively). We can now routinely achieve approximately 1,000-fold of the luciferase signal over the background in the HA-mediated transduction of the target cells (both 293T and A549 cells) by neuraminidase treatment to the producer cells. Thus, the HA(QH)/HIV pseudovirions (see below) used for subsequent experiments were generated using the commercially purchased neuraminidase to treat the 293T producer cells to improve transduction efficiency.
HA-mediated transduction is sensitive to lysosomotropicagents
Multiple human cell lines are susceptible to HA-mediated viral infection
Transduction of cell lines from different species
Name of cell line
3.5 × 102
4.8 × 103
4.8 × 106
2.4 × 106
5.5 × 102
5.9 × 103
3.1 × 106
1.1 × 106
Hu, cervical carcinoma
5.3 × 102
4.1 × 103
1.2 × 107
2.0 × 104
5.4 × 102
2.2 × 103
9.2 × 106
6.0 × 104
5.0 × 102
1.8 × 103
4.6 × 106
3.1 × 104
5.3 × 102
6.5 × 104
1.4 × 107
8.1 × 106
Hu, T lymphocyte
1.0 × 103
1.7 × 103
1.5 × 107
2.1 × 103
6.1 × 102
3.4 × 103
1.7 × 107
3.9 × 104
5.1 × 102
3.5 × 103
1.6 × 107
4.4 × 104
5.1 × 102
3.4 × 103
4.6 × 106
4.1 × 103
6.2 × 102
2.1 × 103
1.6 × 107
1.6 × 103
4.8 × 102
2.0 × 103
4.7 × 106
9.5 × 103
5.4 × 102
1.0 × 103
1.1 × 107
2.7 × 103
6.6 × 102
1.5 × 103
1.7 × 107
1.7 × 103
5.6 × 102
2.2 × 103
1.7 × 107
1.6 × 103
Mouse, B lymphocyte
5.3 × 102
2.4 × 103
2.3 × 104
2.6 × 104
7.0 × 102
3.2 × 103
1.8 × 107
2.3 × 103
The sixteen cell lines can be roughly classified into three different groups, susceptible, moderately susceptible, and resistant to the HA-mediated transduction. The susceptible cells include 293T, A549, and Huh8, all of which were derived from human tissues. These cells, when challenged with HA(QH)/HIV virions, gave luciferase levels of roughly 100–1000-fold higher than the background controls (106-107 RLUs vs 103-104 RLUs with HIV vector alone). HeLa (human), HepG2 (human), SAOS2 (human), Vero E6 (African green monkey), and two avian cell lines (QT6, quail, and DF-1, chicken) are moderately susceptible to the HA-mediated transductions, giving approximately 5–10 fold higher RLUs than the HIV alone controls. The other seven cell lines from different species (CHO, Lec1, COS-7, MDBK, Jurkat, 3T3, and RAW264.7) were resistant to the HA-mediated transductions under the experimental conditions (Table 1).
Human lung cell lines are susceptible to HA-mediatedtransduction
Transduction of different lung cell lines
Name of cell line
2.3 × 103
3.1 × 106
1.7 × 106
1.8 × 103
3.0 × 106
2.1 × 106
7.2 × 103
1.7 × 107
7.6 × 104
2.3 × 103
6.8 × 106
5.1 × 103
1.9 × 103
1.7 × 107
2.5 × 103
Relative levels of cell surface sialic acids do not correlatewith the HA(QH)-mediated entry
Mutational analysis of the H5N1 HA receptor binding domain
The receptor binding domain of hemagglutinin (HA) has been well characterized and studied with respect to key amino acids which are responsible for mediating attachment of the viral glycoprotein to its receptor, sialic acid [28–31]. These studies have defined which amino acids within this domain mediate attachment to 2,3SA as well as what amino acid mutations switch binding to 2,6SA. In the H1 serotypes, the single amino acid change at position 190 from glutamic acid (E) to aspartic acid (D) is crucial for the virus to adapt to usage of the human 2,6SA receptor. For the H2 and H3 serotypes, two amino acid alterations are required. A switch of glutamine (Q) to leucine (L) at position 226 and a switch of glycine (G) to serine(S) at position 228 equates a shift from avian receptor to human receptor specificity. In addition the following residues have been implicated in altering sialic acid binding including H183F andL194A. Further studies specifically targeted towards the HA of the A/Vietnam/1203/2004 H5N1 virus demonstrated the importance of mutation E190D reduced the binding to 2,3SAs, as well as the double mutant Q226L/G228S. Together these studies strongly suggest that residues 190, 226 and 228 are crucial for sialic acid binding and in fact, determine the preference for either 2,3SA or 2,6SA.
To further examine the entry tropism mediated by HA, we generated mutations targeted to residues within the receptor binding domain that have been previously implicated in altering SA binding preference. Using site-directed mutagenesis, we created the following mutations within the H5N1 HA codon optimized backbone: H183F, E190D, L194A, Q226L, G228S, and Q226L/G228S. All mutations were confirmed through sequencing. Western blot analysis of viral particles showed similar levels of HA incorporation for each substitution as well as similar overall levels of viral particle production as measured by p24 levels (data not shown).
Binding analysis of the recombinant HA1 proteins to thetarget cells
The purified proteins were used to evaluate their binding properties to 293T cells which are permissive to the HA-mediated transduction. Neither HA17–268 nor HA89–268, with the amounts of proteins used here, displayed much binding to the target cells, which was measured by flow cytometry (Fig. 5C). In contrast, HA17–340 and especially HA89–340showed a dose-dependent binding to the target cells. To further confirm this finding, 2 μg of each protein was used to bind three cell lines including 293T. As shown in Fig. 5D, HA89–340 displayed binding to both 293T and HeLa cells, both of which are permissible to the HA-mediated transduction, while it did not show any binding to Jurkat cells, a non-permissive cell line. The other three fusion proteins did not give detectable binding under this condition. Thus the following experiment was done using HA89–340.
Different pseudotyping systems have been developed in dissecting the roles of the glycoproteins of highly pathogenic enveloped viruses such as Ebola virus in viral entry and host tropism. To date, such systems have not been used often in entry studies of influenza viruses, although several groups have previously shown the feasibility of this technique in applications such as vaccine development and gene targeting using various viral vectors [32–37]. In this report, we have developed an efficient HIV-based pseudotyping system for avian influenza H5N1 to circumvent the strict requirements in handling this highly pathogenic virus. We anticipate that this system will be invaluable for screening and developing potential entry inhibitors against H5N1. In addition, this pseudotyping system can be easily adapted for entry studies of 1918 Spanish flu and other highly pathogenic influenza viruses with alleviated safety concerns.
Multiple viral factors including HA, NA, the polymerase complex, and NS1 have been implicated in determining the cell and host tropism of influenza viruses [12, 13, 38–42]. Among them, HA is known to be one major determinant which dictates the host restriction [12, 43, 44]. One of the applications of viral pseudotyping systems such as the one developed for H5N1 in this report is to distinguish the host restriction at the entry step determined by HA from those restrictions at the post-entry steps determined by other viral proteins. Here, we demonstrate that several salient features of HA have been recapitulated by the HIV-based pseudotyping system in this report. First, efficient HA-mediated transduction is dependent on neuraminidase treatment on the producer cells (see Results and Fig. 1B), consistent with the notion that NA is involved in cleaving the sialic acid (SA) and thus releasing the budding viral progeny from the cell surface . Furthermore, neuraminidase treatment can be replaced by cotransfection of a human N1 NA gene with the HA gene and HIV vector. It is likely that the HA-mediated transduction efficiency reported in this study, even though very efficient already, can be further optimized by co-expressing M2 in 293T producer cells since others have shown a synergy of M2 and NA in viral particle release . Second, it is well-documented that influenza viruses enter the host cells via receptor-mediated endocytosis in a pH-dependent manner . The sensitivity of HA(QH)-mediated transduction to both lysosomotropic agents demonstrated the strict requirement of low pH for the pseudoviral infection to the target cells (see Fig. 2). Together these results give compelling arguments for using HA/HIV pseudotyping as a simple and reliable surrogate system in elucidating the host tropism of H5N1 and other highly pathogenic influenza viruses at the entry step.
In this report, we have examined the cell tropism of a highly pathogenic avian influenza virus H5N1, which was originally isolated from the infected migratory birds in Qinghai Lake of Western China , at the entry step. The surprising finding is that the most susceptible cell lines for the HA(QH)-mediated transduction are those derived from humans, 293T (kidney) and Huh 8 (liver), and all of the human lung cell lines tested, A549 and NCI-H661 consistent with the recent reports that H5N1 virus can attach to the lower respiratory tract and lung of humans [46, 47]. In stark contrast, two avian cell lines, QT6 (quail fibroblasts) and DF-1 (spontaneously immortalized chicken embryo fibroblasts), were transduced by HA(QH)/HIV virions at much lower frequencies (see Tables 1 and 2). These results indicate that this highly pathogenic H5N1 virus can enter numerous human cells including those derived from human lungs more efficiently than that in the two avian cell lines, suggesting that the HA protein of H5N1 can effectively interact with the cognate cellular receptor(s) on human cells to initiate viral infection. Furthermore it appears that other viral and/or human determinants, rather than HA, restrict efficient transmission of H5N1 to humans in a sustained manner. Therefore we believe that while the H5N1 virus still needs to acquire the ability, either through mutations or genomic re-assortment or both, to produce sustained infection in humans, it appears that this final step for the virus to overcome is at the post-entry level.
Another important implication of the current study is that either an unidentified host co-factor or an alternate linkage of sialic acid may be necessary for mediating H5N1 entry and infection. It is well documented that avian influenza viruses preferentially bind to sialic acid (SA) receptors on avian cells where the SA is predominantly joined to the sugar chain through an α2–3 linkage, while human-adapted influenza viruses have an increased affinity to the SA receptors of the α2–6 linkage , a major form in the human respiratory tract. It is believed that a switch in preferential receptor-binding from the α2–3 linked SA to the α2–6 linked SA by HA protein is a prerequisite for an avian influenza virus to emerge as a pandemic threat in human populations . Recently it was suggested that in addition to the SA linkages, glycan topology as well as sulfation and fucosylation can dictate human adaptation of avian H5N1 virus HA . The question is why the HA protein of H5N1, an avian influenza virus, can transduce human cells more efficiently than non-human cells including avian cells. It is possible that accumulated mutations in the receptor binding domain (RBD) have increased the binding affinity of HA(QH) to the α2–6 linked SA receptors on the human cells. Structural and binding studies of a closely related HA (A/Vietnam/1203/2004, or Viet04 HA) in a recent report are somewhat consistent with this notion. It was found that Viet04 HA is more related to the 1918 and other human HAs than to a 1997 duck H5 HA (DK97 HA) and there are only two noticeable substitutions in the conserved residues of RBDs, E216 and P221 in DK97 HA vs R216 and S221 in Viet04 HA. Viet04 HA was shown to bind α2–6 glycans with some affinity, while DK97 HA did not display any binding . Since the corresponding residues in HA(QH) are K216 and S221 at these positions, it can be assumed that HA(QH), just like Viet04 HA, can bind the α2–6 SA receptors on the human cells to initiate viral entry. However, Viet04 HA was shown to bind to the α2–3 glycans with higher affinity than the α2–6 glycans , which is most likely true for HA(QH). At the same time, HA(QH) is able to mediate more efficient transduction in human cells than avian cells. Further, there is no obvious correlation between the HA-mediated transduction efficiency and the surface levels of 2,3 or 2,6 SAs displayed by several cell lines, and HA substitutions of the SA-binding residues do not greatly impact the HA-mediated transduction or target cell binding. It is possible that an alternative linkage of sialic acid, such as 2,8 or 2,9, may play a role in mediating viral attachment and entry. Here we propose that SA is necessary, but not sufficient to act as the cellular receptor and another surface molecule (or molecules) in addition to SA is also required to mediate efficient H5N1 entry. Indeed it was reported that SA specificity of avian influenza viruses may not restrict initial avian-to-human transmission . Although no such molecule(s) has been identified up to date, a recent report indeed suggests that a host N-linked glycoprotein is required for human influenza virus entry . Therefore, a similar (or different) protein on the human cells such as A549 can act as the co-factor in mediating efficient H5N1 entry, while the same protein on the avian cells is not as efficient as the co-factor either due to a low level of surface expression or low binding affinity to HA. In contrast, the resistant cells such as L2 (rat lung), Lec 1 (Chinese hamster ovary), or Jurkat (human T lymphocyte) may not express a functional homolog on the cell surface. This conclusion is in agreement with a recent report that human tissues lacking the SA receptor can be infected with H5N1 viruses .
Finally, it is important to emphasize that the HIV-based pseudotyping system, together with the numerous susceptible and resistant cell lines and a functional recombinant HA protein reported in this study, will greatly facilitate identification of the co-factor(s) for H5N1 and likely other influenza viruses by genetic and biochemical techniques. Identification and characterization of such host co-factor(s) will have a huge impact on our understanding of the host restriction of H5N1 and on development of potential therapeutics against H5N1 pathogenicity.
Plasmids and cell lines
HA (QH) gene is from a highly pathogenic H5N1 influenza virus in goose (Goose/Qinghai/59/05). HA (QH) and HA (codon optimized) were cloned into pcDNA3 and sequences were confirmed. T4-pMV7 and pc-CCR5 were from the National Institute of Health AIDS Research and Reference Reagent Program. NA (A/PR/8/34) influenza virus (H1N1) in the vector pEF6/V5-His-TOPO was kindly provided by John C Olsen (University of North Carolina, Chapel Hill, USA).
The following cell lines were either purchased from ATCC or provided by other investigators: Vero E6, MDBK, NCI-H661, L2, and Lec1 (ATCC); SAOS-2 and RAW264.7(Bin He, University of Illinois at Chicago), A549 (James Cook, University of Illinois at Chicago), HPAEC (Chinnaswamy Tiruppathi, University of Illinois at Chicago, USA), and DF-1 (Douglas Foster, University of Minnesota, USA). The rest of the cell lines used in the study are maintained in the laboratory. All cell lines were maintained in medium according to the protocols supplemented with 10% FBS and penicillin/streptomycin (50 units/ml).
The rabbit polyclonal antibody ab21297, which recognizes 14 amino acids within residues 100–150 of avian influenza A (H5N1) hemagglutinin protein, was purchased from Abcam Inc. The mouse anti-HIV p24 monoclonal antibody was obtained from the National Institute of Health AIDS Reseach and Reference Reagent Program, which was used to estimate the levels of HIV particle production as measured by p24 levels using Western blotting.
Production of HIV pseudovirions
Human embryonic kidney 293T cells were transiently co-transected with 8 μg hemagglutinin envelope expression plasmid with or without 0.5 μg NA and 10 μg Env-deficient HIV vector (pNL4.3.Luc-R-E- or pNL4-3-GFP-R-E-) in 100-mm plates by a standard Ca3(PO4)2 protocol. Sixteen hours post-transfection, cells were washed by phosphate-buffered saline (PBS) without Ca2+, Mg2+, 10 ml fresh medium was added into each plate. Forty-eight hours post-transfection, the supernatants were collected and filtered through a 0.45-μm-pore size filter (Nalgene) and the pseudovirions were directly used for infection.
Infection assay of HIV pseudovirions
The HIV pseudovirions (0.5 ml/well) prepared above were incubated with various cell types, seeded at 5 × 104/well in 24-well plates. For HIV-Luc virions, the targeted cells were lysed in 50 μl of cell culture lysis reagent (Promega) 48 h post-infection. The luciferase activity was measured with a luciferase assay kit (Promega) and an FB12 luminometer (Berthold detection system) according to the supplier's protocols. The experiments were repeated three times. For HIV-GFP virions infection, the targeted cells were observed by fluorescence microscopy.
The 293T producer cells were treated with a commercial neuraminidase (New England Biolabs) 26 h and 46 h post-transfection at concentrations of 0, 5, 10, 20, 50, 100, 200 units/ml to optimize the yield of HIV pseudovirions, and later on at 100 units/ml for generation of pseudovirions. Pseudovirions were harvested 48 h post-transfection.
Detection of HA incorporation in pseudotyped viruses
To examine incorporation of HA(QH) to HIV particles, 4 ml of the collected supernatant was layered onto a 1.5-ml cushion of 20% sucrose (wt/vol) in PBS and centrifuged at 55,000 rpm for 45 min in a Beckman SW55 rotor. The pelleted HIV virions were lysed in 40 μl of Triton X-100 lysis buffer(50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and a protease inhibitors cocktail consisting of 10 μg of leupeptin per ml, 5 μg of aprotinin per ml, and 2 mM phenylmethylsufonyl fluoride), and a 35 μl sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. The membrane was first incubated with anti-H5N1 HA1 polyclonal antibody ab21297(1:500) for 1 h and then probed with peroxidase-conjugated goat anti-rabbit antiserum (Pierce) for 40 min. The bands were visualized by the chemiluminescence method according to the protocol of the supplier (Pierce). In these experiments, HIV p24 level was determined by Western blotting as an estimate for the relative amounts of the pseudovirions.
NH4Cl and Bafilomycin A1 treatment
Cells were treated with 3.125, 6.25, 12.5, 25, and 50 nM Bifilomycin A1 (BAF) (Sigma) or 3.125, 6.25, 12.5, 25, and 50 mM ammonium chloride (NH4Cl) 30 min prior to infection. For the infection of the HIV envelope (gp120/gp41) pseudotyped HIV virions, 293T cells were first co-transfected with T4-pMV7 and pc-CCR5 36 h prior to infection.
Lectin Binding Assay
5 × 105 cells (293T, A549, NCI-H661, HeLa, L2, CHO, DF1, and QT6) were washed with PBS, pelleted, and resuspended in phosphate-buffered saline. Each cell type was incubated independently with 100 μg/mL of fluorochrome-conjugated lectins, Maackia amurensis agglutinin (MAA)-TRITC and Sambucus nigra agglutinin (SNA)-FITC (EY Laboratories) for 15 minutes in the dark. Cells were then washed 3 times with PBS and analyzed by flow cytometry.
Receptor-binding Domain Mutagenesis
Mutations were generated in the codon optimized HA backbone using a Site-Directed Mutagenesis Kit (Stratagene) and custom primers.
Fusion Protein Production and Purification
To study HA binding to the target cells, plasmids encoding HA fusion protein (different fragment of HA fused to Fc of human IgG) were constructed and the fusion proteins were purified. NheI-BamHI fragment of S1.hIgG was replaced with the PCR product of different fragments of the codon optimized HA gene. Plasmids of HA.hIgG were tranfected into 293T cells using the calcium phosphate method. After overnight, cells were re-fed with protein-free VP-SFM media supplemented with 4 mM L-glutamine (Gibco). Supernatants were collected twice at 48 and 72 hours post-tranfection and filtered through 0.45 μM membrane (Millipore). The supernatants were applied to a column of protein A beads (Santa Cruz Biotechnology) followed by three washing with 10 ml PBS. The proteins were eluted three times with 1 ml 0.1 M glycine (pH 2.8) and immediately neutralized with 60 μl Tris-HCl (pH8.0) each time. Then the proteins were dialyzed in 3.5K Slide-A-Lyzer Dialysis Cassettes (Pierce) and concentrated by centrifuging in YM-10 Centricon (Millipore). The protein concentrations were measured using the BCA Protein Assay Kit (Pierce).
Cells (5 × 105 293T or A549) were blocked in 500 μl volume of PBS/1%BSA on ice for 0.5 hour. Then different amounts of the purified proteins were incubated with cells in 500 μl volume of PBS/1%BSA on ice for 1.5 hour. Cells were incubated with no purified proteins as a negative control. Next the cells were washed twice with PBS/1%BSA and incubated with anti-human antibody conjugated with FITC (1:100 dilution, Sigma) in 500 μl volume of PBS/1%BSA on ice for 40 minutes. Then cells were washed three times with PBS/1%BSA followed by one washing with PBS and subjected to flow cytometry. Relative mean fluorescent intensity (MFI) was calculated by subtracting MFI of the negative control from MFI of cells incubated with different purified proteins.
We thank John Olson, Douglas Foster, Bin He, James Cook, Lili Wang, Chinnaswamy Tiruppathi, and Douglas Foster for cell lines or reagents. The laboratory research was supported by National Institutes of Health grants AI 059570 and CA 092459 (L.R.). Jizhen Wang was supported by a University of Illinois at Chicago Fellowship. H.X. and G.F.G. were partly supported by grants from Ministry of Science & Technology, China (2004BA519A29, 2005CB523001) and National Science Foundation of China (NSFC 30599434, 30525010). J. Y. and Y.C were partly supported by the National Natural Science Foundation of China (30270308, 30599432) and the Ministry of Science and Technology of China (2006CB910103, 2007CB914800).
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