N-Linked Glycosylation Facilitates Sialic Acid-Independent Attachment and Entry of Influenza A Viruses into Cells Expressing DC-SIGN or L-SIGN

CHO Pro-5 cells (62) were obtained from the American Type Culture Collection (ATCC), Manassas, VA. The glycosylation mutant cell line, Lec2, derived from CHO Pro-5 cells (19, 44, 63) was also obtained from the ATCC. Both cell lines were cultured in alpha-minimal essential medium (αMEM; Gibco-BRL, New York) supplemented with 10% (vol/vol) fetal calf serum (JRH Biosciences, Kansas), 4 mM l-glutamine, 100 IU of penicillin, 10 μg of streptomycin/ml, nonessential amino acids (Gibco-BRL), and 50 μM β-mercaptoethanol. LA-4 cells were also obtained from the ATCC and cultured in Kaighn's modification of Ham F-12 medium (Gibco), supplemented as described above.

Transfected Lec2 cells were generated by using neomycin-resistant plasmids encoding human DC-SIGN (46) or human L-SIGN (47). The pcDNA3-DC-SIGN plasmid was obtained through the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH). pcDNA3-DC-SIGN was obtained from S. Pohlmann, F. Baribaud, F. Kirchhoff, and R. W. Doms, and the pcDNA3-L-SIGN plasmid was obtained through the AIDS Research and Reference Program, Division of AIDS, NIAID, NIH. pcDNA3-DC-SIGNR was obtained from S. Pohlman, E. Soilleux, F. Baribaud, and R. W. Doms. Subconfluent Lec2 cells in six-well tissue culture plates (Nunc, New York) were transfected by using FuGene 6 transfection reagent (Roche Diagnostics, Switzerland) according to the manufacturer's instructions. Control-transfected Lec2 cells (designated Lec2-control cells) were generated by using a neomycin-resistant plasmid expressing cytoplasmic hen egg ovalbumin (OVA) (6) generously donated by A. M. Lew and J. L. Brady, The Walter and Eliza Hall Institute, Parkville, Australia. This construct lacks the sequence for cell surface trafficking ensuring intracellular expression of the OVA protein. For the selection of stable transfectants expressing DC-SIGN and L-SIGN or cytoplasmic OVA, transfected cells were cultured in the presence of 1 mg of the selective antibiotic G418 (Gibco)/ml. Clones with high surface expression of DC-SIGN and L-SIGN were obtained via limiting dilution and selected by flow cytometric analysis after staining with monoclonal antibodies (MAbs) directed to human DC-SIGN (clone 120507, conjugated to allophycocyanin [APC]; R&D Systems, Inc.) or human L-SIGN-PE (clone 120604, conjugated to phycoerythrin [PE]; R&D Systems, Inc.). Lec2-DC-SIGN, Lec2-L-SIGN, and Lec2-OVA cells were cultured in supplemented αMEM as described above, in the presence of 1 mg of G418/ml to maintain transgene expression.

The influenza A virus strains used in the present study were A/PR/8/34 (PR8, H1N1) and BJx109 (H3N2), a high-yielding reassortant of PR8 with A/Beijing/353/89 (Beij/89; H3N2) bearing the H3N2 surface glycoproteins. The seasonal H1N1 virus strains A/New Caledonia/20/1999 (New Cal/99) and A/Solomon Islands/3/2006 (Sol Is/06) were obtained from the World Heath Organization Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia. Based on sequence analysis, New Cal/99 and Sol Is/06 both bear four potential sites of N-linked glycosylation on the head of HA (GenBank accession numbers AY289929 and CY031340, respectively). All viruses were grown in 10-day-old embryonated eggs by standard procedures and titrated on Madin-Darby canine kidney (MDCK) cells, and viruses were purified from allantoic fluid by rate zonal sedimentation on 25 to 75% (wt/vol) sucrose gradients, as described previously (2).

Additional reassortant influenza viruses used in the present study were generated by eight-plasmid reverse genetics as previously described (42). The viruses included (i) 7:1 reassortants consisting of the PR8 backbone with either the HA or NA gene from Beij/89 (RG-PR8-Beij/89 HA and RG-PR8-Beij/89 NA, respectively), (ii) eight genes from PR8 (RG-PR8), and (iii) a 6:2 reassortant consisting of six genes from PR8 and the HA and NA from Beij/89 (RG-PR8-Beij/89 HA/NA). The rescued viruses were recovered after 3 days and amplified in the allantoic cavity of 10-day-old embryonated hens' eggs.

Levels of cell surface SA-(α-2,3)-Gal were determined by using the biotinylated plant lectin Maackia amurensis agglutinin II (MAA; EY Laboratories, California), which binds specifically to SA-(α-2,3)-Gal. Cells were detached from plastic flasks using 0.75 mM EDTA in Tris-buffered saline (TBS; 0.05 M Tris-HCl in 0.15 M NaCl [pH 7.4]) and incubated with 5 μg of biotinylated-MAA (b-MAA)/ml in lectin buffer (TBS containing 10 mM CaCl₂ and 1 mg of bovine serum albumin [BSA]/ml; Sigma-Aldrich, Missouri) at 4°C for 30 min. The cells were washed, and bound MAA was detected by using streptavidin conjugated to APC and flow cytometry. To ensure binding specificity, cells were pretreated for 30 min at 37°C with 200 mU of broad-spectrum bacterial sialidase derived from Vibrio cholerae (type III; Sigma Aldrich)/ml to remove SA prior to b-MAA binding.

The ability of influenza virus to bind to cells was determined by using a virus-binding assay. Briefly, detached cells were incubated with 5 μg of purified BJx109 virus/ml in lectin buffer at 4°C for 30 min. Bound virus was detected by using biotinylated MAb recognizing the HA of Beij/89 (MAb C1/1; L. E. Brown, produced in the Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Australia) in conjunction with streptavidin conjugated to APC and flow cytometry. All antibody-binding steps were performed at 4°C in lectin-binding buffer. In experiments assessing the Ca²⁺ dependence of virus binding, CaCl₂ was omitted from the lectin-binding buffer and replaced with 10 mM EDTA.

Binding of mannan to the cell surface was assessed by using an adaptation of the virus-binding assay. Briefly, detached cells were incubated with biotin-labeled mannan (b-mannan; 10 μg/ml) in lectin-binding buffer for 1 h at 4°C. Biotinylated mannan used in the study was prepared as previously described (31). Bound mannan was detected using streptavidin conjugated to APC in conjunction with flow cytometry. In experiments assessing the Ca²⁺ dependence of mannan binding, CaCl₂ was omitted from the lectin-binding buffer and replaced with 10 mM EDTA.

Cells were cultured overnight in eight-well chamber slides (Lab-Tek), and confluent cell monolayers were washed with serum-free αMEM and infected with influenza virus as described previously (49). At 6 to 8 h postinfection, slides were washed in phosphate-buffered saline (PBS), fixed in 80% (vol/vol) acetone, and stained with MAb MP3.10g2.IC7 (WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia), specific for the nucleoprotein (NP) of type A influenza viruses, followed by fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (Dako, Glostrup, Denmark). The percentage of infected cells was determined by costaining with propidium iodide and counting the total number of cells versus FITC-positive cells under ×100 magnification. A minimum of four random fields were selected for counting, assessing at least 200 cells for each sample. Infected cells were photographed by using a Leica DMLB microscope (Leica Microsystems, Germany) and a Leica DFC 490 camera in conjunction with Leica IM50 Image Manager software. In some experiments, cell monolayers were pretreated before infection with 200 mU of bacterial sialidase from Vibrio cholerae type III (Sigma-Aldrich)/ml, Clostridium perfringens (Sigma-Aldrich) or Arthrobacter ureafaciens (Roche), or with 10 mg of mannan (Sigma-Aldrich)/ml in serum-free medium for 1 h at 37°C to remove cell surface SA or block C-type lectins, respectively.

To determine release of infectious virus from cells, confluent cell monolayers were infected in chamber slides as described above. Cell supernatants were collected at 2 and 24 h postinfection, incubated with TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin (10 μg/ml; Sigma-Aldrich) for 45 min at 37°C to facilitate cleavage of the viral HA₀ (56), and titers of infectious virus were determined by plaque assay on MDCK cells.

Viral RNA (vRNA) and mRNA in virus-infected cells were determined by using quantitative real-time reverse transcription-PCR (qRT-PCR). Briefly, 10⁶ cells in 24-well tissue culture plates were infected with 2 × 10⁷ PFU of BJx109 for 1 h at 37°C, washed twice, and incubated in serum-free medium. At 2 and 8 h postinfection, RNA was extracted from cells by using an RNeasy minikit (Qiagen) and stored at −70°C. Levels of matrix (M) gene vRNA and mRNA were determined via qRT-PCR using TaqMan chemistry. The primers and probes for the influenza A virus M gene were as follows: forward primer, 5′-GAC CRA TCC TGT CAC CTC TGA C-3′; reverse primer, 5′-GGG CAT TYT GGA CAA AKC GTC TAC G-3′; and probe, 5′-FAM-TGC AGT CCT CGC TCA CTG GGC ACG-BHQ1 (17). The forward primer was used for vRNA detection, and the reverse primer was used for mRNA detection with the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA) to produce cDNA. cDNA was then used for real-time PCR with TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster City, CA) on an ABI 7500 fast real-time PCR instrument (Applied Biosystems) using a standard program. vRNA and mRNA copy numbers were calculated according to a standard curve generated using 10¹, 10², 10³, 10⁴, and 10⁶ copies of M gene RNA.

Enzyme-linked immunosorbent assay (ELISA) plates were coated overnight with 50 μl of purified influenza virus in PBS and then blocked for 1 h with BSA (10 mg/ml). The wells were washed with PBS containing 0.05% Tween 20 (PBST) and incubated for 2 h with 2 mg of biotin-labeled concanavalin A (ConA; Sigma-Aldrich)/ml in PBS containing 5 mg of BSA per ml. The wells were washed again, and bound lectin was detected after incubation with streptavidin conjugated to horseradish peroxidase (HRP; Silenus, Victoria, Australia). To confirm that different viruses bound to ELISA wells with similar efficiency, additional virus-coated and blocked wells were incubated for 2 h with MAb 165, which recognizes the cross-reactive host antigen common to all egg-grown influenza viruses (50), followed by sheep anti-mouse immunoglobulins conjugated to HRP (Silenus).

Whole-cell lysates were prepared using a buffer comprising 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% (vol/vol) Triton X-100, 1 mM CaCl₂, 1 mM MgCl₂, and broad-spectrum protease inhibitor cocktail (Roche, Manheim, Germany), followed by incubation with cells for 1 h on ice. Lysates were clarified by centrifugation at 10,000 × g for 3 min. Protein concentrations were determined by Bradford assay (Bio-Rad Protein Dye; Bio-Rad, California). Samples (∼10 μg of protein) were boiled for 5 min before separation by SDS-PAGE under nonreducing conditions using 10 to 12.5% gels, followed by transfer to polyvinylidene difluoride (PVDF) membrane (Millipore) in Tris-glycine transfer buffer (25 mM Tris containing 192 mM glycine and 10% [vol/vol] methanol; pH 8.3).

To detect DC-SIGN and L-SIGN in whole-cell lysates by Western blot, membranes were blocked in phosphate-buffered saline (PBS) containing 0.5% (wt/vol) BSA and 0.1% (vol/vol) Tween 20 overnight at 4°C after protein transfer. All subsequent wash and antibody binding steps were performed in PBS containing 0.05% (vol/vol) Tween 20. Membranes were then incubated for 1 h at room temperature with an MAb recognizing both DC- and L-SIGN (clone DC28; R&D Systems, Inc.) that had been labeled with biotin (EZ-link Sulfo-NHS-LC-LC-Biotin; Pierce, Illinois) according to the manufacturer's instructions. Bound MAb was detected using SA-HRP in conjunction with enhanced chemiluminescence (ECL; Western Lightning Plus ECL; Perkin-Elmer, Connecticut). Blots were developed by using the Kodak Image Station 4000Mm, and images were managed by using Adobe Photoshop software.

To detect virus binding, VOPBAs were performed where membranes were blocked with VOPBA buffer (TBS containing 1% [wt/vol] BSA, 0.05% [vol/vol] Tween 20, and 5 mM CaCl₂) for 3 to 4 h at 4°C. Purified BJx109 (2 μg/ml) was incubated overnight, the membrane was washed, and 2 μg of biotinylated MAb C1/1 was added/ml. Bound virus was detected by using streptavidin-conjugated HRP and ECL imaging, as described above. Note that all steps were performed in VOPBA buffer and that antibody binding was performed at 4°C. To assess the Ca²⁺ dependence of virus binding, CaCl₂ was omitted from the VOPBA buffer and replaced with 10 mM EDTA.

Graphing and statistical analysis of data was performed by using GraphPad Prism (GraphPad Software, San Diego, CA). For comparison of multiple data sets, a one-way analysis of variance (ANOVA) with Tukey's multiple comparative analysis was used. A P value of ≤0.05 was considered significant.

Lec2 cells, a mutant CHO cell line, are deficient in terminal SA residues due to a defect in the transport of SA across Golgi vesicles arising from lack of functional CMP-SA transporter (44, 63). Parental CHO cells express SA-(α-2,3)-Gal, but not SA-(α-2,6)-Gal, due to a deficiency in galactoside α-2,6-sialyltransferase (14, 35). Therefore, we used the plant lectin MAA to detect SA-(α-2,3)-Gal on parental CHO Pro5 cells and on Lec2 CHO cells. CHO Pro5 cells bound high levels of MAA, and binding to Lec2 cells was markedly reduced (Fig. 1 A). As expected, desialylation of CHO Pro5 cells with bacterial sialidase led to a marked reduction in MAA binding. Desialylation of the Lec2 cells was also associated with a modest reduction in b-MAA binding (Fig. 1A, upper right panel), which is consistent with studies demonstrating that levels of SA on Lec2 cells are drastically reduced, but not absent, compared to CHO Pro5 cells (36, 63). Lec2 cells were grown in media containing fetal bovine serum, a potential source of gangliosides to cells (23, 34), which may explain the modest binding of b-MAA to the cell surface.

As numerous studies have demonstrated the importance of cell surface SA in mediating influenza virus infection (reviewed by Skehel and Wiley [56]), we investigated the ability of influenza virus to bind to and infect SA-deficient Lec2 cells. Consistent with previous reports (12), influenza virus (strain BJx109) bound efficiently to the surface of CHO Pro5 cells (Fig. 1B), and the cells were susceptible to infection (Fig. 1C). In contrast, BJx109 bound poorly to Lec2 cells (Fig. 1B), and the fluorescence intensity was similar to that of the mock control (i.e., CHO Pro5 cells with no virus added). Moreover, Lec2 cells were largely resistant to infection by BJx109 (6% ± 2% compared to 59% ± 5% infected cells for Lec2 and CHO Pro5 cells, respectively, at the highest inoculum dose tested). The differential ability of BJx109 to infect Lec2 and CHO Pro5 cells was confirmed by using a fluorescence-activated cell sorting (FACS)-based approach where 70% ± 9% of CHO cells expressed intracellular NP at 8 h postinfection compared to 4% ± 2% of Lec2 cells using an inoculum dose of 50 PFU per cell (data not shown; values represent means ± the standard errors of the mean [SEM] from three independent experiments).

To examine the potential of human C-type lectins to act as attachment and/or entry receptors for influenza A virus, we transfected Lec2 CHO cells with human DC-SIGN and L-SIGN and selected for clones showing stable cell surface expression of each receptor as described in Materials and Methods. Flow cytometric analyses confirmed high levels of cell surface expression of either DC-SIGN (Fig. 2A, left panel) or L-SIGN (Fig. 2A, right panel) on Lec2-DC-SIGN and Lec2-L-SIGN cells, respectively, compared to control cells expressing intracellular OVA (Lec2-control).

To confirm the expression of proteins of appropriate molecular size and establish the migration patterns of DC-SIGN and L-SIGN proteins expressed by Lec2 cells, Western blot analysis of total cell lysates prepared from Lec2-control, Lec2-DC-SIGN, or Lec2-L-SIGN cells was performed using a cross-reactive MAb that detects both DC-SIGN and L-SIGN. As seen in Fig. 2B, proteins 49 to 55 kDa in size were detected in lysates from Lec2-DC-SIGN and Lec2-L-SIGN cells but were absent in Lec2-control cells.

DC-SIGN and L-SIGN contain C-type carbohydrate recognition domains that bind to mannose-rich oligosaccharides (21, 40). Lec2 CHO cells are deficient in terminal SA and, based upon the published sequences, DC-SIGN (GenBank accession number M98457) and L-SIGN (GenBank accession number AF245219) are predicted to have one site of N-linked glycosylation that may be sialylated. Since the expression of MMR, a related C-type lectin, in Lec2 cells was associated with major defects in lectin binding and internalization (65), it was critical to determine whether DC-SIGN and L-SIGN expressed on the surface of Lec2 cells retained functional integrity by assessing their ability to bind mannan, a complex plant polysaccharide rich in terminally branched mannose residues. Flow cytometric analysis demonstrated that both Lec2-DC-SIGN (Fig. 3, upper panel) and Lec2 L-SIGN (lower panel), but not Lec2-control cells, bound mannan. Moreover, the presence of EDTA abrogated binding of mannan to Lec2 cells transfected with either DC-SIGN or L-SIGN. Together, these data demonstrate that DC-SIGN and L-SIGN expressed on the surface of Lec2 CHO cells retain C-type lectin activity.

Given that DC-SIGN and L-SIGN expressed on Lec2 cells retained C-type lectin activity, we next assessed the ability of influenza virus to bind to the surface of Lec2-DC-SIGN and Lec2-L-SIGN cells. As seen in Fig. 4 A, BJx109 virus bound to Lec2-DC-SIGN (upper panel) and Lec2-L-SIGN (lower panel) cells but not to Lec2-control cells, and binding was abolished in the presence of EDTA. Binding of influenza virus to DC-SIGN and L-SIGN was also examined by VOPBA, wherein cell lysates of Lec2-DC-SIGN, Lec2-L-SIGN, or Lec2-control cells were separated by SDS-PAGE and probed with BJx109 in the presence of Ca²⁺ (Fig. 4B, left panel) or EDTA (Fig. 4B, right panel). Detection of bound virus revealed interactions with numerous proteins, with particularly intense binding to species of 49 to 55 kDa in lysates from Lec2-DC-SIGN and Lec2-L-SIGN cells. Note that these species show a similar migration pattern to DC-SIGN and L-SIGN detected by Western blotting (Fig. 2B) and that VOBPA failed to detect species of this molecular size in Lec2-control cells. Binding of virus to the 49- to 55-kDa species, but not to other bands detected, was abrogated in the presence of EDTA, a finding consistent with Ca²⁺-dependent binding to C-type lectins. The identity of the other virus-binding bands detected in Lec2 cell lysates was not investigated further but may represent intracellular species, since binding of BJx109 to the surface of Lec2 CHO cells was negligible (Fig. 1B).

SA-deficient Lec2 cells expressing cell surface DC-SIGN or L-SIGN bind influenza virus in a Ca²⁺-dependent manner. We next investigated the ability of DC-SIGN and L-SIGN to rescue the permissivity of Lec2 cells to infection by BJx109 virus. Compared to control Lec2 cells expressing intracellular OVA, expression of either DC-SIGN or L-SIGN was associated with a marked enhancement in susceptibility to infection by BJx109 (Fig. 5A). Virus-infected Lec2-DC-SIGN and Lec2-L-SIGN cells were identified using immunofluorescence to detect nuclear expression of viral NP at 8 h postinfection (Fig. 5B). After exposure of Lec2-DC-SIGN and Lec2-L-SIGN cells to BJx109, viral (v)RNA and messenger (m)RNA were markedly upregulated between 2 and 8 h postinfection, a result indicative of virus entry, genomic replication, and transcription (Fig. 5C). Consistent with immunofluorescence data (Fig. 1C and 5A), vRNA and mRNA were also upregulated in control CHO cells, but not in Lec2-OVA cells. These findings indicate that the poor ability of influenza virus to infect Lec2 cells does not represent a defect in the intrinsic ability of Lec2 cells to support virus replication but rather that Lec2 cells lack appropriate cell surface receptors for virus entry. The levels of infectious virus in supernatants from CHO, Lec2-DC-SIGN, or Lec2-L-SIGN cells exposed to BJx109 did not increase between 2 and 24 h postinfection (Fig. 5D), indicating that infectious progeny virus were not released from infected cells.

To formally address the role of cell surface SA and mannose-specific C-type lectins in the enhanced susceptibility of Lec2 transfectants to virus infection, cells were either (i) treated with bacterial sialidase prior to infection or (ii) incubated with mannan, a multivalent inhibitor of DC-SIGN and L-SIGN, prior to and during infection with BJx109. As seen in Fig. 6i, BJx109 infected the parental cell line CHO Pro5 to high levels, and treatment with bacterial sialidase reduced infection to <5%, whereas the addition of mannan did not alter susceptibility to infection, consistent with SA-dependent virus entry. Lec2-control cells were largely resistant to infection (Fig. 6ii), and expression of either DC-SIGN (Fig. 6iii) or L-SIGN (Fig. 6iv) restored susceptibility to infection. In contrast to CHO Pro5 cells, inclusion of 10 mg of mannan/ml led to a marked reduction in susceptibility of Lec2-DC-SIGN and Lec2-L-SIGN to infection by BJx109.

Lec2 cells have markedly reduced levels of cell surface SA due to a mutation in the CMP-SA transporter; however, it is possible that this mutation may allow partial activity (36), enabling low-level SA expression. Therefore, it was important to determine any possible contribution of cell surface SA to the enhanced susceptibility of Lec2-DC-SIGN and Lec2-L-SIGN to virus infection. In contrast to CHO Pro5 cells (Fig. 6i), treatment of Lec2-DC-SIGN or Lec2-L-SIGN cells with bacterial sialidase did not reduce susceptibility to infection by BJx109 and was, in fact, generally associated with a modest enhancement in infection. Data are shown for cells treated with Vibrio cholerae (type III) sialidase, and similar results were obtained with an equivalent concentration of sialidase from either Clostridium perfringens or Arthrobacter ureafaciens (data not shown). Together, these data indicate that DC-SIGN- and L-SIGN-mediated infection of Lec2 cells can occur via lectin-mediated interactions in the absence of cell surface SA.

DC-SIGN and L-SIGN recognize mannose-rich oligosaccharides (21, 40), and influenza viruses are known to differ markedly in the glycosylation of HA and NA glycoproteins. In particular, virus strains differ in the degree of glycosylation on the globular head of HA (56), and this has been shown to modulate sensitivity to soluble C-type lectins (50, 51) and the MMR (49, 73). BJx109 (H3N2) has four potential sites of N-linked glycosylation on the globular head of the HA compared to PR8 (H1N1), which has none. BJx109 and PR8 bear three and two N glycosylation sites on the NA head, respectively (predictions were made by sequence analysis with the NetNGyc 1.0 Server). BJx109 virus is a high-yielding reassortant of PR8 with A/Beijing/353/89 (H3N2), bearing the H3N2 glycoproteins. ELISA wells coated with increasing concentrations of purified BJx109 and PR8 were probed with ConA, a plant lectin that binds high-mannose and hybrid-type oligosaccharides. BJx109 bound ConA more efficiently than PR8, and binding was inhibited in the presence of mannan (Fig. 7 A). MAb 165, a carbohydrate-specific MAb that recognizes the cross-reactive host antigen common to all egg-grown influenza viruses (50), was used to confirm equivalent coating levels of virus (data not shown). Periodate treatment of BJx109 or PR8 to oxidize carbohydrate moieties expressed on HA/NA also abrogated ConA binding (Fig. 7A). Together, these data demonstrate that BJx109 expresses high levels of high-mannose and hybrid glycans, whereas PR8 does not.

To gain insight into the relative importance of HA and NA as ligands for DC-SIGN/L-SIGN, we utilized additional reassortant viruses engineered by reverse genetics (RG). Thus, either the HA or the NA of Beij/89 was inserted into the seven-gene backbone of PR8 by RG to generate RG-PR8-Beij/89 HA or RG-PR8-Beij/89 NA, respectively. In these experiments, PR8 was also rederived by RG (RG-PR8), as well as a virus bearing internal components of PR8 with Beij/89 HA and NA (RG-PR8-Beij/89 HA/NA). As seen in Fig. 7B, all RG viruses infected CHO Pro5 cells to equivalent levels (panel i) and were poor in their ability to infect Lec2-control cells (panel ii). RG-PR8-Beij/89 HA/NA infected Lec2-DC-SIGN (panel iii) and Lec2-L-SIGN cells (panel iv) to high levels, whereas RG-PR8 was particularly poor in its ability to infect Lec2 cells expressing either C-type lectin. Of interest, the RG virus bearing the Beij/89 HA (RG-PR8-Beij/89 HA) infected Lec2-DC-SIGN and Lec2-L-SIGN cells to levels equivalent to that of RG-PR8-Beij/89 HA/NA. In contrast, RG-PR8-Beij/89 NA infected Lec2 cells expressing either human C-type lectin very poorly. Together, these data indicate that expression of the HA glycoprotein of Beij/89 is associated with efficient infection by Lec2 cells expressing either DC-SIGN or L-SIGN.

The PR8 (H1N1) virus was particularly poor in its ability to infect Lec2 cells expressing DC-SIGN or L-SIGN, likely due to the low levels of glycosylation on the viral HA. In contrast, viruses bearing the highly glycosylated HA of Beij/89 (H3) infected cells efficiently. To confirm that the poor ability of PR8 to infect transfected cells was not a general feature of H1 subtype viruses, we used two additional virus strains, New Cal/99 and Sol Is/06, both of which bear four potential glycosylation sites on the head of HA. As seen in Fig. 7C, both viruses infected parental CHO Pro5 cells to equivalent levels (panel i) and were poor in their ability to infect Lec2-OVA cells (panel ii), although Sol Is/06 was somewhat more efficient than New Cal/99. In contrast, Lec2-DC-SIGN (panel iii) and Lec2-L-SIGN cells (panel iv) were both susceptible to infection by either New Cal/99 or Sol Is/06 viruses. Thus, the poor ability of PR8 to infect Lec2 cells via DC-SIGN or L-SIGN is not a general property of H1N1 subtype viruses. More likely it relates to the low levels of mannose-rich glycans expressed by this virus (Fig. 7A).