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. ​(Fig.1B).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

To test pH-dependence of the HA-mediated transduction, the sensitivity of HA/HIV virions to lysosomotropic agents was examined on 293T target cells. The target cells were first treated with either ammonium chloride (NH₄Cl) or Bafilomycin A1 at different concentrations (see Materials and Methods for the details) prior to incubation with the HA(QH)/HIV pseudovirions, and the infected cells were assayed for luciferase activity 48 hours post-infection. As shown in Fig. ​Fig.2,2, both NH₄Cl and Bafilomycin A1 could greatly inhibit the HA(QH)-mediated transduction efficiency. As controls, the HIV glycoprotein gp120/gp41-mediated transduction, which was previously shown to be pH-independent[24,25], was not adversely affected by either compounds, while the Ebola GP-mediated transduction, a pH-dependent virus[19,21], was severely inhibited by both compounds. These results are consistent with the notion that entry of H5N1, like that of other influenza viruses[10,26], is pH-dependent.

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Figure 2

Sensitivity of the HA-mediated transduction to lysosomotropic agents. The 293T target cells were first treated with either ammonium chloride (NH₄Cl) or Bafilomycin A1 (BAF) at various concentrations prior to the challenge by the HA(QH)/HIV, Ebola GP/HIV, and HIVgp120/41/HIV (labeled as GP120/HIV here) pseudovirions. The luciferase activities of the infected cells were determined 48 h post-infection. The data of the NH₄Cl treatment at the concentration of 3.125 mM and the Bafilomycin treatment at the concentration of 3.125 nM are shown here. Values are means of triplicate samples ± SD.

Multiple human cell lines are susceptible to HA-mediated viral infection

To characterize and compare the host tropism at the entry step for H5N1, the HA(QH)/HIV pseudovirions were incubated with numerous cell lines derived from different species and tissues and the transduction efficiencies were measured as the relative levels of luciferase activity (RLUs) in the challenged cells (Table ​(Table1).1). In these experiments, the VSV-G-mediated HIV pseudovirion transduction was used as a positive control since VSV-G displays a very broad host range. Among the 17 cell lines examined (Table ​(Table1),1), all were highly susceptible to VSV-G pseudovirus infection as indicated by greater than 10⁶ RLUs with the only exception being A20, a mouse B lymphocytes-derived cell line, which gave only approximately 10-fold higher luciferase activity when challenged with the VSV-G/HIV (2.3 × 10⁴ RLUs) over the HIV vector alone (2.4 × 10³ RLUs), suggesting a possible post-entry block for HIV in this cell line. The remaining sixteen cell lines gave high luciferase activities of more than 6 logs of RLUs when challenged with the VSV-G/HIV virions, ranging from about 100- to more than 1000-fold over the background levels (cell alone or HIV vector alone), indicating there is no post-entry block for HIV infection in these cells. Thus, the relative levels of RLUs of these cells challenged with the HA(QH)/HIV virions indicate the transduction efficiencies of the cells by the HA protein.

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 ​(Table11).

Human lung cell lines are susceptible to HA-mediatedtransduction

To investigate whether the lung cells were susceptible to HA-mediated transduction, we obtained and tested three more lung cell lines in addition to A549 cells: NCI-H661(human), HPAEC (human), and L2 (rat), and the results are summarized in Table ​Table2.2. Transduction efficiencies are representative of repeated experiments displaying similar trends. In these experiments, Lec 1 cells were used as a negative control since previously Lec 1 was shown to be resistant to influenza viral entry by others[27] and us in this report (see Table ​Table1).1). Like A549, NCI-H661 could be efficiently transduced by HA(QH)/HIV (100–1,000 fold higher of RLUs than the background). HPAEC could be transduced at a lower efficiency (more than 10-fold higher of RLUs than the background). In contrast, the rat lung L2 cells were relatively resistant to transduction by HA(QH). It should be pointed out that all of these results were visually confirmed using an HIV vector carrying a GFP as the reporter gene under a microscope (data not shown). These results demonstrated that human lung cells, but not rat lung cells, are susceptible to HA-mediated transduction.

Relative levels of cell surface sialic acids do not correlatewith the HA(QH)-mediated entry

To better understand the role SA plays in transduction mediated by HA(QH), we sought to determine the overall relative abundance of 2,3 and 2,6SA linkages on 13 cell lines previously tested in the transduction assay, however glycan topology nor sulfation or fucosylation were able to be assessed using this system. Cell lines were chosen based on their species and tissue origin as well as on transduction levels. Utilizing fluorescently conjugated lectins which bind specifically to either 2,3SA (MAA TRITC) or 2,6SA (SNA-I FITC), equal number of cells were incubated with each lectin for 15 minutes in the dark. Cells were then washed 3 times with PBS, resuspended, and analyzed via FACS to determine relative sialic acid levels. 293T (human kidney), A549 (human lung), NCI-H661 (human lung), HeLa (human cervical carcinoma), Huh8 (human liver), and Jurkat (human T lymphocyte) cell lines all demonstrated high levels of the human influenza receptor, 2,6SA, at levels of 700–1,600 with NCI-H661 cells having a modest level of more than 400, while CHO (Chinese hamster ovary), DF1 (chicken embryo), and QT6 (quail fibroblast) cell lines all had at least 5-fold lower levels (Fig. ​(Fig.3).3). DF1, NIH 3T3, Jurkat, and A549 cells had greater levels of 2,3SA compared to the other cells tested. A comparison between transduction levels and either 2,3SA or 2,6SA reveals that there is no correlation between either receptor level or the level of entry. The human lung cell line, A549, and the HeLa cell line have similar profiles for both 2,3SA and 2,6SA, however A549 cells are susceptible to H5N1 HA-mediated transduction while HeLa cells are not, suggesting that the overall level of either 2,3SA or 2,6SA does not correlate with H5N1-HA mediated transduction. Thus, we do not see a correlation between the levels of either 2,3, or 2,6 SA receptor and the HA-mediated transduction, suggesting a possible role of a co-factor in H5N1 entry either in conjunction with or perhaps independently of sialic acid.

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Figure 3

Analysis of relative surface levels of 2,3 and 2,6 sialic acid. Relative surface levels of 2,3 and 2,6 sialic acid in different cell lines as measured using the fluorescently conjugated lectins 2,3 sialic acid specific MAA-TRITC and 2,6 sialic acid specific SNA-I-FITC. Data is representative of repeated experiments displaying similar trends. Values are means of triplicate samples ± SD.

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).

Viral pseudotypes were generated as previously mentioned, however, co-transfection of the neuraminidase gene, (A/Puerto Rico/8/34(H1N1)), along with our luciferase reporter and HA gene, allowed viral egress to occur and alleviated the need to treat producer cells with exogenous purified neuraminidase. Target cell lines, 293T (data not shown) and A549, had comparable levels of 2,3SA and 2,6SA, suggesting that mutations generated in the HA protein should affect viral entry in these cell lines in a similar manner. Each HA pseudovirus with or without any substitutions gave luciferase levels of 10⁶–10⁷ units while still remaining within the linear range of the assay. The mutation E190D which has been shown to decrease affinity of the H5N1 HA for 2,3SA did not alter transduction of either cell line dramatically as represented by RLUs in A549 cells (Fig. ​(Fig.4B).4B). Mutations Q226L/G228S which have also been implicated in decreasing affinity towards 2,3SA showed little affect in infectivity. None of the other mutations implicated in altering the binding properties of hemagglutinin for sialic acid showed a dramatic difference in transduction levels either. These results indicate that these substitutions did not greatly alter the HA-mediated transduction, consistent with the notion that a cofactor may be involved in H5N1 entry or that an alternate linkage of sialic acid is necessary.

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Figure 4

Effect of hemagglutinin receptor-binding domain mutations in viral entry. (A) Sequence alignment of mutations generated. (B) Relative infectivity of pseudoviruses containing specified receptor-binding domain substitutions as measured by luciferase activity. Data is representative of repeated experiments displaying similar trends. Values are means of triplicate samples ± SD.

Antibodies

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.

Neuraminidase treatment

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.

Receptor-binding Domain Mutagenesis

Mutations were generated in the codon optimized HA backbone using a Site-Directed Mutagenesis Kit (Stratagene) and custom primers.