Adenovirus type 37 uses sialic acid as a cellular receptor

Abstract

Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) alpha2, have recently been identified. In the absence of CAR, MHC-I alpha2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expressing CHO cells and MHC-I alpha2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via alpha(2-->3)-linked sialic acid saccharides rather than alpha(2-->6)-linked ones, since (i) alpha(2-->3)-specific but not alpha(2-->6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with alpha(2-->3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of alpha(2-->3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I alpha2.

FIG. 1

Binding of ³H-labeled Ad5 or Ad37 virions to CHO cells expressing CAR (CHO-CAR) or human α2 integrin (CHO alpha2). Pelleted cells (2 × 10⁵) were resuspended in 100 μl of BB containing Ad5 or Ad37 virions. Cells were incubated on ice for 1 h, and nonbound virions were washed away. Cell-associated radioactivity was counted as described in Materials and Methods.

FIG. 2

(A and B) Binding of ³H-labeled Ad37 (A) or Ad5 (B) virions to 2 × 10⁵ V. cholerae neuraminidase-treated A549 (⧫), CAR-expressing CHO (■), HCE (●), or E8.1 (∗) cells. (C) Binding of labeled Ad37 to 2 × 10⁵ Pro-5 cells premixed with different concentrations of WGA. Error bars show standard errors.

FIG. 3

(A) Expression of sialic acid on sialic acid-lacking Lec2 cells and Pro-5 cells (parental cells for Lec2 cells), which express sialic acid, as measured by use of FITC-labeled WGA in a FACS assay. (B) Binding of ³H-labeled Ad37 or Ad5 to Pro-5 cells or to Lec2 cells. Cell-associated radioactivity was counted as described in Materials and Methods.

FIG. 4

Infectivity of Ad5, Ad19p, or Ad37 in 2 × 10⁵ A549 cells in 24-well plates. Prior to infection, the cells were incubated at 37°C for 1 h with different concentrations of V. cholerae neuraminidase diluted in PBS containing 2% FCS. Virus stocks were added in dilutions corresponding to 5,000 FFU/well and incubated for 1 h at 37°C. Nonbound virions were washed away. At 40 h postinfection, the cells were stained and examined as described in Materials and Methods.

FIG. 5

Ad37 binding to Pro-5 cells pretreated with different proteases. Pelleted Pro-5 cells (10⁶) were resuspended in PBS containing 0, 200, or 2,000 mU of protease and incubated at 37°C. The proteases bromelain (⧫), V8 protease (▴), and ficin (■) were used. After 1 h, the proteases were washed away, and the cells were pelleted. Cells were resuspended in 100 μl of BB containing ³H-labeled Ad37. After 1 h of incubation on ice, nonbound virions were washed away. Cell-associated radioactivity was counted as described in Materials and Methods. Error bars show standard errors.

FIG. 6

Ad37 binds to Pro-5 cells via (2→3)-linked sialic acids, and the binding is inhibited by GpA but not by aGpA. (A) ³H-labeled Ad37 virions were incubated on ice in 100 μl of BB with 10 μg of GpA or asialo-GpA (aGpA) per ml. After 1 h, the mixtures were added to 2 × 10⁵ pelleted Pro-5 cells. The resuspended cells were incubated on ice for 1 h. Alternatively, 2 × 10⁵ Pro-5 cells were mixed with 100 g of lectin from M. amurensis [MA, specific for (2→3)-linked sialic acid], S. nigra [SN, specific for (2→6)-linked sialic acid], or WGA per ml. After 1 h on ice, ³H-labeled Ad37 virions were added to make a final volume of 100 μl, and the mixtures were incubated on ice for 1 h. Nonbound virions were washed away, and cell-associated radioactivity was measured as described in Materials and Methods. CTRL, control. (B) Pro-5 cells (2 × 10⁵) were incubated at 37°C with different concentrations of (2-3)-specific neuraminidase. After 1 h, the neuraminidase was washed away, and the pelleted cells were resuspended in 100 μl of BB containing ³H-labeled Ad37 virions. The mixtures were incubated on ice for 1 h. Nonbound virions were washed away, and cell-associated radioactivity was measured as described in Materials and Methods. Error bars show standard errors.