Receptor usage of a newly emergent adenovirus type 14

Abstract

Recently, cases of severe respiratory illness in military and civilian populations have been associated with a new genomic variant of adenovirus (Ad) serotype 14, designated Ad14a. Compared to the Ad14 reference strain (de Wit), this new virus had a deletion of two amino acid residues in the fiber protein knob. Here we tested whether this mutation changed receptor usage of Ad14a compared to Ad14-de Wit. Competition studies with radio-labeled viruses revealed that both Ad14-de Wit and Ad14a used the same receptor which is hitherto unknown. We also found that recombinant fiber knobs only partially blocked attachment of Ad14a, indicating that virus capsid proteins other than the fiber are involved in infection.

Fig. 1. Comparison of amino acid sequence of Ad14-de Wit, Ad14a, and species B Ads 3, 7, 11, and 35

The β-sheets are marked by a box. The mutation in Ad14a is marked by an arrow. The Genbank accession numbers were AAW33140 (Ad14-de Wit); P35774 (ad11p); AAF14128 (Ad7a); AAA66361 (Ad35p); CAA26029 Ad3.)

Fig. 2. Competition of viruses for attachment on 293 cells

A and B) 293 cells were pre-incubated with Ad14-de Wit, Ad14a, Ad3, Ad35 or 10μg/ml CD46 antibody (MEM-258, Serotec) for one hour on ice. After washing, ³H-Ad14-de Wit, (Fig 2A, left panel), ³H-Ad14a, (Fig. 2A, right panel), ³H-Ad3, (Fig. 2B, left panel) or ³H-Ad35 (Fig. 2B, right panel) was added for another hour on ice. After washing, cell-associated radioactivity was determined. N=3. Bars indicate the means. Standard deviation was less than 10% in all cases. C) Affinity of Ad14-de Wit and Ad14a to 293 cells. To generate Scatchard blots, cells were incubated with increasing MOIs of ³H-Ad14-de Wit or ³H-Ad14a and the number of cells associated viral particles was measured after one hour of incubation on ice. The y-axis shows the ratio between bound particles to total input particles minus bound particles. The x-axis shows the number of bound particles. The binding affinities (K_a) of virus were calculated on the basis of the slope with standard Excel software as described previously (Tuve et al., 2006).

Fig. 3. Fiber knob competition Ad14-de Wit and Ad14a attachment

A) Analysis of recombinant Ad fiber knobs. Purified Ad35, Ad14-de Wit, and Ad14a knob protein (1μg/lane) was run as native protein (N) or after denaturation (D) on a polyacrylamide gel. Coomassie brilliant blue staining (left panel) revealed the trimeric knob form, which is converted into monomers after boiling. Western blots (right panel) were analyzed for CD46 binding to fiber knobs by subsequent incubation with sCD46, anti-CD46 Mab, and anti-mouse IgG-HRP. B) Fiber knob competition. Cells were pre-incubated with 0.4μg or 4μg knob protein on ice for 1 hour. Then ³H-Ad14-de Wit or ³H-Ad14a virus was added and cell- associated radioactivity was measured after 1 hour incubation. N=3. Bars indicate the means. Standard deviation was less than 10% in all cases.

Fig. 4. Internalization of Ad particles

A) Cells were detached from culture dishes by incubation with versene, washed with PBS, and chilled on ice for 45 minutes. A total of 2 × 10⁵ cells per tube were resuspended in 150μl of ice-cold adhesion buffer containing 8,000VP/cell of ³H-labeled wild-type Ad14-de Wit or Ad14a virus. After 60 minutes of incubation on ice, cells were washed with PBS to remove unbound virus, one set of samples were incubate with trypsin at 37°C for 20 minutes (w/Trysin) the other sample with PBS (w/o Trypsin). Cells were washed and cell associated radioactivity was measured. B) Cells were incubated with ³H-Ad14-de Wit or ³H-Ad14a virus for 10 minutes on ice and then at 37°C for the indicated time. Non-internalized virus particles were removed by 20 minutes of trypsin treatment of cells and cell-associated radioactivity was measured. The Y-axis shows the percentage of viral particles internalized compared to the number of viral particles bound per cell. (N=3)