PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Virology. Author manuscript; available in PMC 2010 September 30.
Published in final edited form as:
PMCID: PMC2743766
NIHMSID: NIHMS127123

Bovine adenovirus serotype 3 utilizes sialic acid as a cellular receptor for virus entry

Abstract

Bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) entry into the host cells is independent of Coxsackievirus -adenovirus receptor and integrins. The role of sialic acid in BAd3 and PAd3 entry was investigated. Removal of sialic acid by neuraminidase, or blocking sialic acid by wheat germ agglutinin lectin significantly inhibited BAd3, but not PAd3, transduction of Madin Darby bovine kidney cells. Maackia amurensis agglutinin or Sambucus nigra (elder) agglutinin treatment efficiently blocked BAd3 transduction suggesting that BAd3 utilized α(2,3)-linked and α(2,6)-linked sialic acid as a cell receptor. BAd3 transduction of MDBK cells was sensitive to sodium periodate, bromelain, or trypsin treatment indicating that the receptor sialoconjugate was a glycoprotein rather than a ganglioside. To determine sialic acid-containing cell membrane proteins that bind to BAd3, virus overlay protein binding assay (VOPBA) was performed and showed that approximately 97 and 34 kDa cell membrane proteins containing sialic acid bind to BAd3. The results suggest sialic acid serves as a primary receptor for BAd3.

Keywords: adenovirus receptor, bovine adenovirus serotype 3, porcine adenovirus serotype 3, sialic acid, virus attachment, lectin, neuraminidase

INTRODUCTION

Adenovirus (Ad) entry into the host cells is initiated by attachment of the viral particle with a primary receptor on the cell surface followed by interactions with a secondary receptor leading to virus endocytosis into a clathrin-coated vesicle and subsequent transportation to the endosome (Berk, 2007). Acidification of the endosome leads to partial disassembly of the capsid followed by transport of the viral genome into the host cell nucleus. For subgroup C human Ad (HAd) such as HAd serotype 5 (HAd5), the primary cell receptor is Coxsackievirus adenovirus receptor (CAR), an immunoglobulin superfamily molecule shared as a receptor by Coxsackieviruses and Ads (Bergelson et al., 1997). The interaction between the viral penton base and the secondary receptor αv integrins is essential for HAd5 internalization into the host cells. In addition to CAR, several other cell surface molecules have been identified as primary or secondary receptors involved in attachment or internalization of many other Ads (Sharma et al., 2009; Zhang and Bergelson, 2005). These receptor molecules include CD46 (Gaggar et al., 2003; Segerman et al., 2003), CD80 (B7-1)/CD86 (B7-2) (Short et al., 2004), major histocompatibility complex class I (MHC I) (Hong et al., 1997), vascular cell adhesion molecule-1 (VCAM-1) (Chu et al., 2001), heparan sulphate proteoglycans (HSPGs) (Dechecchi et al., 2000) and sialic acid (Arnberg et al., 2000a).

A variety of nonhuman Ads have been proposed as promising gene delivery vectors (Bangari and Mittal, 2006). Bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) belong to the genus Mastadenovirus of family Adenoviridae and are isolated from a normal calf or pig, respectively. The transcriptional map of BAd3 or PAd3 is similar to that of HAd5 (Reddy et al., 1998a; Reddy et al., 1998b). We have previously demonstrated that BAd3 or PAd3 entry is independent of CAR and integrins, and that BAd3, PAd3 and HAd5 cellular receptors are distinct (Bangari et al., 2005c; Bangari and Mittal, 2005).

A number of viruses including some serotypes of HAds have been shown to utilize sialic acid molecules as a cellular receptor (Lehmann et al., 2006a). Subgroup D Ads, HAd8, HAd19a and HAd37, which are frequently associated with epidemic keratoconjunctivitis (Gordon et al., 1996; Kemp et al., 1983), utilize sialic acid as a cell receptor (Arnberg et al., 1997; Arnberg et al., 2000a; Lehmann et al., 2006d). In this manuscript, we endeavor to determine whether sialic acid plays a role in BAd3 or PAd3 attachment and internalization. Our experiments involve the evaluation of BAd3 or PAd3 vector [expressing the green fluorescent protein (GFP) gene] transduction of Madin-Darby bovine kidney (MDBK) cells pretreated with 1) neuraminidase to remove cell surface sialic acid molecules, 2) wheat germ agglutinin lectin (WGA) to block availability of sialic acid molecules, 3) sialic acid linkage-specific lectins for competitive inhibition of Ad transduction, and 4) with either sodium periodate to remove sialic acid-conjugated carbohydrates or proteases to remove sialic acid-conjugated proteins. Our results provide strong evidence for sialic acid as a component of BAd3 receptor, whereas PAd3 entry was independent of sialic acid. Furthermore to find out sialic acid-containing cell membrane proteins that attach to BAd3, virus overlay protein binding assay (VOPBA) was conducted. Sialic acid-containing cell membrane proteins of approximately 97 and 34 kDa demonstrated association to BAd3.

RESULTS

Enzymatic removal of cell surface sialic acid inhibits BAd3 transduction

To investigate the role of sialic acid in BAd3 or PAd3 entry, we used MDBK cells for transduction assays. The presence of sialic acid on the MDBK cell surface was confirmed by labeling the cells with a fluorescent dye, tetramethyl rhodamine isothiocyanate (TRITC)-conjugated wheat germ agglutinin (WGA) from Triticum vulgaris that binds to sialic acid. To examine the effect of neuraminidase treatment on the surface sialic acid molecules, untreated and neuraminidase-treated MDBK cells were examined by staining with TRITC-conjugated WGA. As shown in Fig. 1A, the specific cell membrane staining was observed in untreated cells. In neuraminidase-treated cells, the circular membrane staining was diminished compared to the untreated cells indicating that MDBK cells have sialic acid molecules on the surface and neuraminidase treatment was effective in the removal of these molecules (Fig. 1B).

Fig.1
Sialic acid on MDBK cell surface is sensitive to neuraminidase treatment

In order to examine if BAd3 or PAd3 utilizes sialic acid as a cell receptor, MDBK cells were treated with neuraminidase. For viral transduction experiments, BAd-GFP, a replication-defective BAd3 vector expressing the GFP reporter gene (Bangari et al., 2005e) and PAd-GFP, a replication-defective PAd3 vector expressing GFP (Bangari and Mittal, 2004c) were used in these experiments. As a control, EGFPNAd5F37, a HAd5 vector with a hybrid fiber knob from HAd37 and the enhanced green fluorescent gene (EGFP) expression cassette (Arnberg et al., 2000a; Cashman et al., 2004a), was included in our experiments. EGFPNAd5F37 has been shown to bind to the sialic acid receptor (Cashman et al., 2004b). HAd-GFP, a HAd5 vector expressing GFP was used as a negative control. As expected, treatment of MDBK cells with neuraminidase inhibited transduction of EGFPNAd5F37 by 43% compared to the untreated group (Fig. 2). Interestingly, pretreatment of MDBK cells with neuraminidase at concentrations of 0.1, 1, or 10 mU/ml reduced BAd-GFP transduction by 87%, 93% and 88%, respectively. No inhibition in transduction by HAd-GFP or PAd-GFP was observed indicating that the cell surface sialic acid was not involved in PAd3 or HAd5 entry.

Fig.2
Neuraminidase treatment of MDBK cells inhibits transduction by BAd3 vector

Pretreatment with sialic acid-binding lectin WGA blocks BAd3 transduction

To further confirm the role of sialic acid in BAd3 entry, a sialic acid-binding molecule, WGA lectin, was used to treat MDBK cells prior to virus transduction. Preincubation of MDBK cells with WGA demonstrated a dose-dependent inhibition in BAd-GFP transduction. BAd-GFP transduction was reduced approximately 97% when the cells were preincubated with 0.05 mg/ml or higher concentrations of WGA (Fig. 3). There was no strong inhibition in the transduction efficiency of EGFPNAd5F37 in cells treated with WGA (Fig. 3). This may be due to the utilization of other potential receptor/s by this chimeric vector. WGA preincubation did not block the attachment of HAd-GFP and PAd-GFP to MDBK cells (Fig. 3). These results further highlighted the importance of cell surface sialic acid in BAd3 entry.

Fig.3
Pretreatment of MDBK cells with WGA lectin inhibits BAd3 transduction vector

Our results with MDBK cells treated either with neuraminidase or WGA lectin clearly showed that PAd3 entry into the host cells was independent of sialic acid molecules on the cell surface. Therefore, additional experiments were directed to further explore the nature of sialic acid molecules that are involved in BAd3 entry.

Both α(2,3)-linked and α(2,6)-linked sialylated glycoconjugates are involved in BAd3 binding

Tissue tropism of some of the Ads is closely related with interaction between viral fiber knob domain and linkage-specific sialic acid receptors. HAd37 has been shown to predominantly bind to α(2,3)-linked sialic acid rather than α(2,6)-linked sialic acid (Arnberg et al., 2000a). Identification of α(2,3)-linked or α(2,6)-linked sialic acid molecules on the cell surface in the lower or upper respiratory tract, respectively in humans (van Riel et al., 2007) certainly holds considerable importance for adenoviral vectors and their tropism for gene delivery.

To further analyze the nature of the sialic acid receptor involved in BAd3 entry, linkage-specific lectins were utilized to block the cell surface sialic acid molecules prior to transduction with EGFPNAd5F37 or BAd-GFP. It is known that Maackia amurensis agglutinin (MAA) attaches to α(2,3)-linked sialic acid; while Sambucus nigra agglutinin (SNA) binds to α(2, 6)-linked sialic acid (Varki and Schauer, 1999). In the present study, MDBK cells were treated with either MAA or SNA prior to transduction with EGFPNAd5F37 or BAd-GFP and at 48 h post-transduction, cells were harvested and analyzed by flow cytometry. There was a dose-dependent inhibition in transduction with EGFPNAd5F37 when cells were pretreated with MAA or SNA (Fig. 4); however, the reduction in transduction was more pronounced with the MAA treatment group. In the case of BAd-GFP, there was significant decline in transduction of cells pretreated with either MAA or SNA (Fig. 4). Pretreatment of MDBK cells with 0.5 mg/ml of MAA or SNA resulted in a drop in BAd-GFP transduction by 90% or 59%, respectively. It appears that BAd3 utilizes α(2,3)-linked sialic acid and α(2,6)-linked sialic acid as a receptor for virus attachment with a preference for α(2,3)-linked sialic acid.

Fig.4
BAd3 utilizes both α(2,3)-linked and α(2,6)-linked sialic acid as a cellular receptor

Sialylated carbohydrate is involved in BAd3 binding

It has been shown that sodium periodate (NaIO4) can destroy carbohydrate moieties by oxidation of vicinal hydroxyl groups on sugars to dialdehydes at acidic pH without altering protein or lipid structures (Stevenson et al., 2004c). To further examine the role of cell surface carbohydrate in BAd3 binding, MDBK cells were pretreated with NaIO4 followed by transduction with either EGFPNAd5F37 or BAd-GFP. Pretreatment of MDBK cells with NaIO4 at a concentration of 1 mM reduced BAd-GFP transduction by 70% compared to the untreated group (Fig. 5) suggesting that sialic acid-containing carbohydrate is a component of the BAd3 receptor. As a control, a 62% reduction in EGFPNAd5F37 transduction was observed in the group treated with NaIO4 at a concentration of 1 mM compared to the untreated group (Fig. 5).

Fig.5
BAd3-interacting sialic acid-containing cell surface molecule contains carbohydrate moieties

Sialic acid molecules recognized by BAd3 is localized on glycoproteins rather than gangliosides

It is known that sialic acid saccharides are expressed on both glycoproteins and glycolipids (gangliosides). To examine if BAd3 virus binding to cells is sensitive to protease treatment, MDBK cells were treated with trypsin or bromelain prior to transduction with EGFPNAd5F37 or BAd-GFP. As shown in Fig. 6, trypsin treatment of MDBK cells reduced BAd-GFP transduction by 81% while bromelain treatment reduced BAd-GFP binding by 30% compared to the untreated group suggesting that the sialic acid-containing BAd3 receptor has a protein component. As expected, approximately a 63% or 78% reduction in EGFPNAd5F37 transduction was observed in trypsin- or bromelain-treated groups, respectively, compared to the untreated groups (Fig. 6).

Fig.6
Sialic-acid containing receptor for BAd3 is sensitive to protease treatment

Neuraminidase treatment blocks BAd3 binding to MDBK cellular membrane protein

VOPBA is a standard technique to identify cell molecules involved in virus binding. We observed that neuraminidase treatment of MDBK cells resulted in reduced transduction of BAd-GFP. To examine whether neuraminidase treatment of MDBK cells affects BAd3 binding to membrane proteins, we performed a VOPBA assay using cell membrane fractions from neuraminidase-treated or untreated MDBK cells. As shown in Fig.7, a prominent and a faint bands of approximately 97 kDa and 34 kDa, respectively (lane 2) were observed in the untreated MDBK cell membrane protein sample, which were absent in the membrane sample obtained from neuraminidase-treated cells (lane 1) or the untreated cell membrane protein samples without the virus overlay (lanes 3 and 4). It appears that the removal of sialic acid molecules from 97 kDa and 34 kDa proteins resulted in the loss of BAd3 binding. Either or both of these two proteins represent sialic acid-containing potential receptor candidates for BAd3. Further characterization of these two proteins will be carried out in future studies.

Fig.7
BAd3 receptor is associated with approximately 97 and/or 34 kDa neuraminidase-sensitive proteins

DISCUSSION

A variety of Ad vectors derived from human or nonhuman species have shown distinct advantages as gene delivery vehicles for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral proteins. Receptor usage by viruses is a major determinant of their tissue tropism, an important consideration for the design of safe and effective gene transfer vectors. The primary attachment is followed by interactions between the virus and a secondary receptor which triggers virus entry (Lehmann et al., 2006c).

Cell entry by BAd3 and PAd3 has been reported to be independent of the HAd receptors, CAR and integrins, and BAd3 and PAd3 receptors are distinct from the HAd5 receptor (Bangari et al., 2005b; Bangari and Mittal, 2004a; Bangari and Mittal, 2005). In this study, we have presented a number of lines of evidence indicating that sialic acid serves as a primary cellular receptor for BAd3 entry. Sialic acid molecules were removed from MDBK cell surfaces with neuraminidase treatment, which led to a dose-dependent reduction in BAd3 transduction. Since virus and cell receptor interaction experiments were performed on ice, it was expected that cell surface sialic acid moieties were involved in the initial stage (attachment) of BAd3 entry. Competitive inhibition assay with a sialic acid-binding lectin, WGA, further confirmed that sialic acid molecules were utilized as a cell receptor for BAd3.

Use of neuraminidase treatment demonstrated the utilization of sialic acid as a receptor for HAd37 (Arnberg et al., 2000a). Sialic acid serves as a cellular receptor for some of the subgroup D HAds (HAd8, HAd19a and HAd37) (Arnberg et al., 2000a; Arnberg et al., 2000b; Arnberg et al., 2002). Neuraminidase or WGA treatment of cells prior to vector transduction clearly demonstrated that sialic acid did not serve as a receptor for PAd3. More work is needed to identify the primary receptor for PAd3 entry into susceptible cells.

The discrepancy in transduction levels of EGFPNAd5F37 in cells treated with neuraminidase or WGA may be partly due to the involvement of other potential receptors since it is a HAd5-based chimeric vector carrying a HAd37 fiber knob. Ad attachment can occur following direct interaction between the penton base and cell surface integrins independent of the fiber knob-primary receptor interactions (Huang et al., 1996b). Fiber-deficient HAd2 virions can infect CAR-negative monocytic cells by a mechanism that involves attachment to integrins αMβ2 and αLβ2, followed by an interaction with αv integrins which is needed for internalization (Huang et al., 1996a).

MAA and SNA lectins have the ability to specifically bind α(2,3)-linked sialic acid or α(2,6)-linked sialic acid, respectively (Lehmann et al., 2006b). It has been shown that HAd37 can specifically bind to α(2,3)-linked sialic acid rather than α(2,6)-linked sialic acid (Arnberg et al., 2000a). Moreover, it has been demonstrated that MAA rather than SNA treatment can significantly reduce EGFPNAd5F37 binding to Chang C cells and human lung carcinoma (A549) cells (Cashman et al., 2004c). Our results with EGFPNAd5F37 in cells pretreated with MAA or SNA were consistent with previous observations. However, in our experiment, both MAA and SNA significantly blocked BAd3 transduction of MDBK cells suggesting that BAd3 virus utilizes both α(2,3)-linked sialic acid and α(2,6)-linked sialic acid as a cell receptor with a slight preference for α(2,3)-linked sialic acid.

Sodium periodate has the ability to destroy carbohydrate moieties without altering the protein or lipid structures (Stevenson et al., 2004b). In our study, MDBK cells pretreated with NaIO4 showed that the sialic-acid-containing BAd3 receptor has a carbohydrate component. Additional results with trypsin or bromelain treatment demonstrated that BAd3 receptor contains a protein component with terminal sialic acid. Similarly, HAd37 binding to Chang C (Wu et al., 2001b) and CHO cells (Arnberg et al., 2000a) was reduced when these cells were pretreated with general or specific proteases. This phenomenon was also observed for EGFPNAd5F37 binding to protease pretreated Chang C and A549 cells (Cashman et al., 2004d). VOPBA further demonstrated that approximately 97 and/or 34 kDa cell membrane proteins with sialic acid molecules may serve as receptor/s for BAd3. Further work is needed to characterize these cell membrane proteins.

Differences in the receptor usage by diverse Ad serotypes provide the distinctive opportunity to exploit the natural diversity of Ad tropism in designing safe and efficient vectors for various gene therapy applications. Sialic acid-binding HAds or chimeric HAd5 vectors with fiber knob derived from subgroup D HAds result in transduction of cell types, such as hematopoietic cells including dendritic cells, which are refractory to transduction by CAR-utilizing HAd vectors (Horvath and Weber, 1988; Rea et al., 2001). The observation that BAd3 utilizes both α(2,3)-linked and α(2,6)-linked sialic acid as a cell receptor will further widen the repertoire of Ad receptors and will be of significance in unraveling the complexity of Ad tissue tropism.

MATERIAL AND METHODS

Cells and viruses

MDBK cells were grown as monolayer cultures in minimum essential medium (MEM) with 10% FetalClone III (Hyclone of Thermo Fisher Scientific Inc., Waltham, MA) and gentamicin (50 µg/ml). Cells were harvested with phosphate-buffered saline (PBS)-EDTA (10 mM sodium phosphate buffer, 150 mM NaCl, and 2 mM EDTA, pH 7.4) to keep the cell receptors intact. Suspended cells were washed twice with binding buffer (BB: PBS with 1% bovine serum albumin, BSA) to remove EDTA. Treated or untreated MDBK cells were transduced with either BAd-GFP, PAd-GFP, HAd-GFP or EGFPNAd5F37 and 2 × 105 cells were seeded per well of a 24-well plate in triplicate and incubated for 48 h in a CO2 incubator in MEM containing 10% FetalClone III.

Construction and propagation of BAd-GFP, PAd-GFP, and HAd-GFP have been described previously (Bangari et al., 2005a; Bangari and Mittal, 2004d). BAd-GFP, PAd-GFP, and HAd-GFP were grown in FBRT-HE1 [fetal bovine retinal cells that express HAd5 E1] (van Olphen et al., 2002), FPRT-E1–5 [fetal porcine retinal cells that express HAd5 E1] (Bangari and Mittal, 2004b) and 293 cell line [human embryonic kidney cell line that expresses HAd5 E1 proteins] (Graham et al., 1977), respectively. The chimeric vector, EGFPNAd5F37 (Cashman et al., 2004e) that contains the HAd5 capsid with fiber knob domain from HAd37 was kindly provided by Dr. R. Kumar-Singh, Department of Ophthalmology and Visual Sciences, and Human Genetics, University of Utah, Salt Lake City, UT) and was propagated in 293 cells . Wild type BAd3 was propagated in MDBK cells as previously described (Bangari et al., 2005d). Since BAd-GFP, PAd-GFP, HAd-GFP, or EGFPNAd5F37 has variable transduction efficiency in MDBK cells, various dilutions of Ad vector were used to obtain transduction efficacy of approximately 25–30%. To obtain this efficiency for BAd-GFP, PAd-GFP, HAd-GFP, or EGFPNAd5F37, a multiplicity of infection (m.o.i.) of 0.5, 1.0, 3.5, and 0.5 tissue culture infectious dose 50 (TCID50) per cell was used.

At 48 h post-transduction, cells were trypsinized, washed once with PBS and fixed with 2% paraformaldehyde, and the percentage of GFP-expressing cells was determined by flow cytometry (BD FACS CantoII, BD Bioscience, San Jose, CA). All experiments were carried out in triplicate. Negative and positive controls were kept for each experiment to make sure the cells used in the experiments were competent for virus uptake.

Wheat germ agglutinin (WGA) staining

MDBK cells in monolayer cultures in 8-well chamber slides (Nalge Nunc International, Naperville, IL) were treated with neuraminidase at a concentration of 1 mU/ml at 37°C for 2 h. Subsequently, cells were washed with PBS twice and then stained with 150 × diluted TRITC-conjugated WGA (E. Y. Laboratories Inc., San Mateo, CA) at room temperature for 15 min. Stained cells were examined under a fluorescent microscope and images were captured at 40× magnification using same setting for treatment and control groups. MDBK cell monolayers without neuraminidase treatment were kept as a positive control.

Neuraminidase treatment

A total of 2 × 105 MDBK cells were treated with Vibrio cholerae neuraminidase (Sigma-Aldrich, St. Louis MO) at concentrations of 0, 0.01, 0.1, 1.0 or 10 mU/ml in BB and incubated for 2 h at 37°C. Subsequently, cells were washed with BB and suspended in 100 µl of BB and kept on ice for 10 min. Treated or untreated MDBK cells were transduced either with BAd-GFP, PAd-GFP, HAd-GFP, or EGFPNAd5F37 on ice for 1 h. Cells washed with MEM with 10% FetalClone III were seeded in 24-well plates and incubated in a CO2 incubator in MEM containing 10% FetalClone III. At 48 h post-transduction, cells were trypsinized, washed once with PBS and fixed with 2% paraformaldehyde and the percentage of GFP-expressing cells was determined by flow cytometry. All experiments were carried out in triplicate.

WGA blocking assay

A total of 2 × 105 MDBK cells were incubated with 100 µl of WGA lectin (Sigma-Aldrich) at concentrations of 0, 0.01, 0.05, 0.1, or 0.5 mg/ml on ice for 1 h. WGA-treated or untreated cells were mixed with various Ad vectors and kept on ice for 1 h. The rest of the procedure was the same as described above for neuraminidase treatment.

Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA) treatment

A total of 2 × 105 MDBK cells were incubated with 100 µl of MAA (Vector Labs, Burlingame, CA) or SNA (Sigma-Aldrich) at concentrations of 0, 0.01, 0.05, 0.1, or 0.5 mg/ml in BB and incubated on ice for 1 h. MAA/SNA treated or untreated MDBK cells were mixed with BAd-GFP or EGFPNAd5F37 and kept on ice for 1 h. The rest of the procedure was the same as described above for neuraminidase treatment. The negative controls (Mock-infected) had a background level around 0.2–0.4 percent and the positive controls (HAd-GFP-infected) showed that approximately 25–30 percent cells expressed GFP, suggesting that the cells used in the experiments were competent for virus uptake.

Sodium periodate (NaIO4) treatment

A total of 2 × 105 MDBK cells were incubated with 100 µl of periodate (Acros Organics, Morris Plains, NJ) at concentrations of 0, 0.01, or 0.1 mM in PBS at room temperature for 30 min. The unreacted periodate was neutralized by adding two volumes of 0.22% (v/v) glycerol in PBS (Stevenson et al., 2004a). Treated or untreated cells were washed twice with BB, kept on ice and mixed with BAd-GFP or EGFPNAd5F37 and incubated on ice for 1 h. The rest of the procedure was the same as described above for neuraminidase treatment. The negative controls (Mock-infected) had a background level around 0.2–0.4 percent and the positive controls (HAd-GFP-infected) showed that approximately 25–30 percent cells expressed GFP, suggesting that the cells used in the experiments were competent for virus uptake.

Protease treatment

A total of 2 × 105 MDBK cells were incubated with 100 µl of trypsin (Invitrogen, Life Technologies Corp., Carlsbad, CA) at concentrations of 0, 50, or 500 µg/ml or with 100 µl of bromelain (Sigma-Aldrich) at concentrations of 0, 200, or 2000 mU/ml in PBS at 37°C for 30 min. Cells were then washed twice with BB, kept on ice and mixed with BAd-GFP or EGFPNAd5F37 and incubated for 1 h. The rest of the procedure was the same as described above for neuraminidase treatment.

Virus overlay protein binding assay (VOPBA)

In order to investigate whether neuraminidase treatment of MDBK cells affects BAd3 binding to cell membrane proteins, a VOPBA assay was carried out using cell membrane fractions from neuraminidase-treated or untreated MDBK cells. Fourteen 150 mm plates of MDBK cells at approximate 90% confluence were treated with PBS-EDTA and washed twice with BB. Cells from seven plates were treated with 1 mU/ml neuraminidase in BB, and cells from the remaining seven plates were mock-treated with BB at 37°C for 2 h and washed twice with cold PBS. Neuraminidase-treated or untreated cells were used for the extraction of membrane proteins as previously described (Wu et al., 2001a). Briefly, cells were resuspended in the membrane solution [250 mM sucrose, 1mM EDTA, 20mM HEPES, pH 7, with protease inhibitors cocktail including Phenylmethylsulfonyl Fluoride and Aprotinin (Sigma-Aldrich)] and incubated on ice for 10 min. This was followed by homogenization with Dounce homogenizer and centrifugation at 3,500 rpm to remove the cellular debris. The supernatant was centrifuged at 35,000 rpm for 1 h and the pellet was resuspended in 0.01M Tris, pH 8.0 containing protease inhibitors and used for VOPBA. Membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane. Non-specific binding was blocked by 5% skimmed milk powder solution at room temperature for 1 h. The blots were overlaid with wild type BAd3 virus at a concentration of 10 µg/ml or were mock-treated without virus. The washed blots were then incubated with 1:1000 diluted rabbit anti-BAd3 polyclonal serum (Moffatt et al., 2000) at room temperature for 1 h. The washed blots were then incubated with 1:2000 diluted horse radish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin IgG (H+L) (Bio-Rad, Hercules, CA) at room temperature for 1 h. The presence of the bound virus was detected by chemiluminescence detection system-SuperSignalR West Pico (Thermo Fisher Scientific Inc), and digital images were captured using a Kodak Image Station (Kodak, Rochester, NY). Efficiency of neuraminidase treatment of MDBK cells that were used for VOPBA was monitored by evaluating inhibition of BAd-GFP transduction.

ACKNOWLEDGEMENTS

We are thankful to Dr. R. Kumar-Singh, Department of Ophthalmology and Visual Sciences, and Human Genetics, University of Utah, Salt Lake City, UT, for providing EGFPNAd5F37 and Jane Kovach for her excellent secretarial assistance. This work was supported by Public Health Service grant CA110176 from the National Cancer Institute.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reference List

  • Arnberg N, Edlund K, Kidd AH, Wadell G. Adenovirus type 37 uses sialic acid as a cellular receptor. J.Virol. 2000a;74:42–48. [PMC free article] [PubMed]
  • Arnberg N, Kidd AH, Edlund K, Olfat F, Wadell G. Initial interactions of subgenus D adenoviruses with A549 cellular receptors: sialic acid versus alpha(v) integrins. J.Virol. 2000b;74:7691–7693. [PMC free article] [PubMed]
  • Arnberg N, Mei Y, Wadell G. Fiber genes of adenoviruses with tropism for the eye and the genital tract. Virology. 1997;227:239–244. [PubMed]
  • Arnberg N, Pring-Akerblom P, Wadell G. Adenovirus type 37 uses sialic acid as a cellular receptor on Chang C cells. J.Virol. 2002;76:8834–8841. [PMC free article] [PubMed]
  • Bangari DS, Mittal SK. Porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture. Virus Res. 2004a;105:127–136. [PubMed]
  • Bangari DS, Mittal SK. Porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture. Virus Res. 2004b;105:127–136. [PubMed]
  • Bangari DS, Mittal SK. Porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture. Virus Res. 2004c;105:127–136. [PubMed]
  • Bangari DS, Mittal SK. Porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture. Virus Res. 2004d;105:127–136. [PubMed]
  • Bangari DS, Mittal SK. Porcine adenovirus serotype 3 internalization is independent of CAR and alpha(v)beta(3) or alpha(v)beta(5) integrin. Virology. 2005;332:157–166. [PubMed]
  • Bangari DS, Mittal SK. Development of nonhuman adenoviruses as vaccine vectors. Vaccine. 2006;24:849–862. [PMC free article] [PubMed]
  • Bangari DS, Sharma A, Mittal SK. Bovine adenovirus type 3 internalization is independent of primary receptors of human adenovirus type 5 and porcine adenovirus type 3. Biochem.Biophys.Res.Commun. 2005a;331:1478–1484. [PMC free article] [PubMed]
  • Bangari DS, Sharma A, Mittal SK. Bovine adenovirus type 3 internalization is independent of primary receptors of human adenovirus type 5 and porcine adenovirus type 3. Biochem.Biophys.Res.Commun. 2005b;331:1478–1484. [PMC free article] [PubMed]
  • Bangari DS, Sharma A, Mittal SK. Bovine adenovirus type 3 internalization is independent of primary receptors of human adenovirus type 5 and porcine adenovirus type 3. Biochem.Biophys.Res.Commun. 2005c;331:1478–1484. [PMC free article] [PubMed]
  • Bangari DS, Shukla S, Mittal SK. Comparative transduction efficiencies of human and nonhuman adenoviral vectors in human, murine, bovine, and porcine cells in culture. Biochem.Biophys.Res.Commun. 2005d;327:960–966. [PubMed]
  • Bangari DS, Shukla S, Mittal SK. Comparative transduction efficiencies of human and nonhuman adenoviral vectors in human, murine, bovine, and porcine cells in culture. Biochem.Biophys.Res.Commun. 2005e;327:960–966. [PubMed]
  • Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science. 1997;275:1320–1323. [PubMed]
  • Berk AJ. Adenoviridae: The Viruses and Their Replication. In: Knipe DM, Howley PM, editors. Fields Virology. Philadelphia: Lippincott Williams & Wilkins; 2007. pp. 2355–2394.
  • Cashman SM, Morris DJ, Kumar-Singh R. Adenovirus type 5 pseudotyped with adenovirus type 37 fiber uses sialic acid as a cellular receptor. Virology. 2004e;324:129–139. [PubMed]
  • Cashman SM, Morris DJ, Kumar-Singh R. Adenovirus type 5 pseudotyped with adenovirus type 37 fiber uses sialic acid as a cellular receptor. Virology. 2004a;324:129–139. [PubMed]
  • Cashman SM, Morris DJ, Kumar-Singh R. Adenovirus type 5 pseudotyped with adenovirus type 37 fiber uses sialic acid as a cellular receptor. Virology. 2004b;324:129–139. [PubMed]
  • Cashman SM, Morris DJ, Kumar-Singh R. Adenovirus type 5 pseudotyped with adenovirus type 37 fiber uses sialic acid as a cellular receptor. Virology. 2004c;324:129–139. [PubMed]
  • Cashman SM, Morris DJ, Kumar-Singh R. Adenovirus type 5 pseudotyped with adenovirus type 37 fiber uses sialic acid as a cellular receptor. Virology. 2004d;324:129–139. [PubMed]
  • Chu Y, Heistad D, Cybulsky MI, Davidson BL. Vascular cell adhesion molecule-1 augments adenovirus-mediated gene transfer. Arterioscler.Thromb.Vasc.Biol. 2001;21:238–242. [PubMed]
  • Dechecchi MC, Tamanini A, Bonizzato A, Cabrini G. Heparan sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions. Virology. 2000;268:382–390. [PubMed]
  • Gaggar A, Shayakhmetov DM, Lieber A. CD46 is a cellular receptor for group B adenoviruses. Nat.Med. 2003;9:1408–1412. [PubMed]
  • Gordon YJ, raullo-Cruz TP, Johnson YF, Romanowski EG, Kinchington PR. Isolation of human adenovirus type 5 variants resistant to the antiviral cidofovir. Invest.Ophthalmol.Vis.Sci. 1996;37:2774–2778. [PubMed]
  • Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J.Gen.Virol. 1977;36:59–74. [PubMed]
  • Hong SS, Karayan L, Tournier J, Curiel DT, Boulanger PA. Adenovirus type 5 fiber knob binds to MHC class I alpha2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO J. 1997;16:2294–2306. [PubMed]
  • Horvath J, Weber JM. Nonpermissivity of human peripheral blood lymphocytes to adenovirus type 2 infection. J.Virol. 1988;62:341–345. [PMC free article] [PubMed]
  • Huang S, Kamata T, Takada Y, Ruggeri ZM, Nemerow GR. Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells. J.Virol. 1996b;70:4502–4508. [PMC free article] [PubMed]
  • Huang S, Kamata T, Takada Y, Ruggeri ZM, Nemerow GR. Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells. J.Virol. 1996a;70:4502–4508. [PMC free article] [PubMed]
  • Kemp MC, Hierholzer JC, Cabradilla CP, Obijeski JF. The changing etiology of epidemic keratoconjunctivitis: antigenic and restriction enzyme analyses of adenovirus types 19 and 37 isolated over a 10-year period. J.Infect.Dis. 1983;148:24–33. [PubMed]
  • Lehmann F, Tiralongo E, Tiralongo J. Sialic acid-specific lectins: occurrence, specificity and function. Cell Mol.Life Sci. 2006b;63:1331–1354. [PubMed]
  • Lehmann F, Tiralongo E, Tiralongo J. Sialic acid-specific lectins: occurrence, specificity and function. Cell Mol.Life Sci. 2006a;63:1331–1354. [PubMed]
  • Lehmann F, Tiralongo E, Tiralongo J. Sialic acid-specific lectins: occurrence, specificity and function. Cell Mol.Life Sci. 2006d;63:1331–1354. [PubMed]
  • Lehmann F, Tiralongo E, Tiralongo J. Sialic acid-specific lectins: occurrence, specificity and function. Cell Mol.Life Sci. 2006c;63:1331–1354. [PubMed]
  • Moffatt S, Hays J, HogenEsch H, Mittal SK. Circumvention of vector-specific neutralizing antibody response by alternating use of human and non-human adenoviruses: implications in gene therapy. Virology. 2000;272:159–167. [PubMed]
  • Rea D, Havenga MJ, van Den AM, Sutmuller RP, Lemckert A, Hoeben RC, Bout A, Melief CJ, Offringa R. Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells. J.Immunol. 2001;166:5236–5244. [PubMed]
  • Reddy PS, Idamakanti N, Song JY, Lee JB, Hyun BH, Park JH, Cha SH, Bae YT, Tikoo SK, Babiuk LA. Nucleotide sequence and transcription map of porcine adenovirus type 3. Virology. 1998a;251:414–426. [PubMed]
  • Reddy PS, Idamakanti N, Zakhartchouk AN, Baxi MK, Lee JB, Pyne C, Babiuk LA, Tikoo SK. Nucleotide sequence, genome organization, and transcription map of bovine adenovirus type 3. J.Virol. 1998b;72:1394–1402. [PMC free article] [PubMed]
  • Segerman A, Atkinson JP, Marttila M, Dennerquist V, Wadell G, Arnberg N. Adenovirus type 11 uses CD46 as a cellular receptor. J.Virol. 2003;77:9183–9191. [PMC free article] [PubMed]
  • Sharma A, Li X, Bangari DS, Mittal SK. Adenovirus receptors and their implications in gene delivery. Virus Res. 2009 in press. [PMC free article] [PubMed]
  • Short JJ, Pereboev AV, Kawakami Y, Vasu C, Holterman MJ, Curiel DT. Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. Virology. 2004;322:349–359. [PubMed]
  • Stevenson RA, Huang JA, Studdert MJ, Hartley CA. Sialic acid acts as a receptor for equine rhinitis A virus binding and infection. J.Gen.Virol. 2004b;85:2535–2543. [PubMed]
  • Stevenson RA, Huang JA, Studdert MJ, Hartley CA. Sialic acid acts as a receptor for equine rhinitis A virus binding and infection. J.Gen.Virol. 2004c;85:2535–2543. [PubMed]
  • Stevenson RA, Huang JA, Studdert MJ, Hartley CA. Sialic acid acts as a receptor for equine rhinitis A virus binding and infection. J.Gen.Virol. 2004a;85:2535–2543. [PubMed]
  • van Olphen AL, Tikoo SK, Mittal SK. Characterization of bovine adenovirus type 3 E1 proteins and isolation of E1-expressing cell lines. Virology. 2002;295:108–118. [PubMed]
  • van Riel D, Munster VJ, de WE, Rimmelzwaan GF, Fouchier RA, Osterhaus AD, Kuiken T. Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals. Am.J.Pathol. 2007;171:1215–1223. [PubMed]
  • Varki A, Schauer R. Sialic Acids. In: Varki A, Cummings RD, Esko JE, Freeze H, Hart GW, Marth J, editors. Essentials of Glycobiology. New York: Cold Spring Harbor Laboratory Press; 1999.
  • Wu E, Fernandez J, Fleck SK, Von Seggern DJ, Huang S, Nemerow GR. A 50-kDa membrane protein mediates sialic acid-independent binding and infection of conjunctival cells by adenovirus type 37. Virology. 2001b;279:78–89. [PubMed]
  • Wu E, Fernandez J, Fleck SK, Von Seggern DJ, Huang S, Nemerow GR. A 50-kDa membrane protein mediates sialic acid-independent binding and infection of conjunctival cells by adenovirus type 37. Virology. 2001a;279:78–89. [PubMed]
  • Zhang Y, Bergelson JM. Adenovirus receptors. J.Virol. 2005;79:12125–12131. [PMC free article] [PubMed]