Dependence of adenovirus infectivity on length of the fiber shaft domain

Abstract

One of the objectives in adenovirus (Ad) vector development is to target gene delivery to specific cell types. Major attention has been given to modification of the Ad fiber knob, which is thought to determine virus tropism. However, among the human Ad serotypes with different tissue tropisms, not only the knob but also the length of the fiber shaft domain varies significantly. In this study we attempted to delineate the role of fiber length in coxsackievirus-adenovirus receptor (CAR)- and non-CAR-mediated infection. A series of Ad serotype 5 (Ad5) capsid-based vectors containing long or short fibers with knob domains derived from Ad5, Ad9, or Ad35 was constructed and tested in adsorption, internalization, and transduction studies. For Ad5 or Ad9 knob-possessing vectors, a long-shafted fiber was critical for efficient adsorption/internalization and transduction of CAR/alphav integrin-expressing cells. Ad5 capids containing short CAR-recognizing fibers were affected in cell adsorption and infection. In contrast, for the chimeric vectors possessing Ad35 knobs, which enter cells by a CAR/alphav integrin-independent pathway, fiber shaft length had no significant influence on binding or infectibility on tested cells. The weak attachment of short-shafted Ad5 or Ad9 knob-possessing vectors seems to be causally associated with a charge-dependent repulsion between Ad5 capsid and acidic cell surface proteins. The differences between short- and long-shafted vectors in attachment or infection were abrogated by preincubation of cells with polycations. This study demonstrates that the fiber-CAR interaction is not the sole determinant for tropism of Ad vectors containing chimeric fibers. CAR- and alphav integrin-mediated infections are influenced by other factors, including the length of the fiber shaft.

FIG. 1

Schematic representation of chimeric fiber proteins incorporated into Ad5 capsids (A) and structure of chimeric fiber genes (B). (A) The Ad fiber can be divided into three domains. The conserved N-terminal tail contains the sequences responsible for association with the penton base. The long shafts of Ad5L, Ad5/9L, and Ad5/35L contain 22 β sheets (37 nm); the short shafts in Ad5S and Ad5/9S contain 8 β sheets (11 nm); Ad5/35S contains 6 β sheets (9 nm). The C-terminal globular knob domains are derived from Ad5 (Ad5L or -S), Ad9 (Ad5/9L or -S), or Ad35 (Ad5/35L or -S) wild-type viruses. Both Ad5 and Ad9 fibers bind to CAR, whereas Ad35 fiber interacts with an unidentified receptor different from CAR. (B) To construct the corresponding chimeric fiber genes, the indicated sets of primers were used to amplify DNA corresponding to fiber domains derived from different serotypes. To amplify DNA encoding the Ad5 fiber tail domain or the Ad5 fiber gene polyadenylation signal, L1 and L2 primers or R1 and R2 primers, respectively, were used. To amplify DNA encoding the fiber shaft domains, the corresponding shaft forward (SF) and shaft reverse (SR) primers were used. To amplify DNA encoding for the fiber knob domains, the corresponding knob forward (KF) and knob reverse (KR) primers were used. The amplified fiber domains were conjoined with a second PCR step employing primers L1 and R2 as described in Materials and Methods.

FIG. 2

Expression of CAR and αv integrins on 293, K562, and Y79 cells. The level of CAR or αv integrin expression on test cells was determined by flow cytometry analysis. 293, Y79, and K562 cells were incubated with anti-CAR (RmcB), anti-αv integrin (L230), or anti-BrdU (as a negative control) primary antibodies as described in Materials and Methods. The binding of primary antibody was developed with anti-mouse IgG-FITC secondary antibody and subsequent flow cytometry analysis for positive staining. The thick, thin, and dotted lines show positive staining with anti-CAR, anti-αv integrin, and anti-BrdU antibodies, respectively. Data shown represent average results of quadruplicate analyses performed on 10⁴ cells. Note that preincubation with the control antibody or with antibody dilution buffer only gave the same intensity of staining (data not shown).

FIG. 3

Attachment and internalization of Ad vectors with modified fibers to 293, K562, and Y79 cells (A) and Southern blot analysis of Ad genomes attached to 293 cells (B). (A) Equal amounts of [³H]thymidine-labeled virions were allowed to attach to () or be internalized into (□) cells as described in Materials and Methods. Cells were then washed, and the number of viral particles bound per cell was determined. The data were obtained from two to four independent experiments performed in triplicate. (B) To analyze the level of Ad attachment, equal amounts of indicated Ad vectors at an MOI of 8,000 genomes per cell were mixed with 3.5 × 10⁵ 293 cells in 100 μl of adhesion buffer and then allowed to attach for 1 h on ice. Then virus-containing medium was removed; cells were washed twice with ice-cold PBS and resuspended in 100 μl of adhesion buffer. Then 100 μl of lysis buffer was added to the cells, and DNA was extracted as described earlier (61) (Virus attachment). To estimate the amount of Ad loaded per sample, the same volume of corresponding Ad as used in the attachment study was mixed with 3.5 × 10⁵ 293 cells in 100 μl of adhesion buffer. Immediately after mixing, 100 μl of lysis buffer was added to cells, and DNA was extracted (Virus load). After purification, DNA concentration was measured spectrophotometrically, and 1 μg of each sample was applied on the agarose gel. Shown are the ethidium bromide-stained 1% agarose gel before blotting, demonstrating that similar amounts of genomic DNA were loaded (top), and the result of Southern blot hybridization of the transferred DNA with a ³²P-labeled 8-kb HindIII fragment of the Ad5 genome, corresponding to the E2 region (bottom). The conditions of DNA transfer and hybridization were described earlier (61). M, molecular weight marker. Arrows indicate the Ad DNA (bottom) or mixture of Ad and cellular DNA (top).

FIG. 3

Attachment and internalization of Ad vectors with modified fibers to 293, K562, and Y79 cells (A) and Southern blot analysis of Ad genomes attached to 293 cells (B). (A) Equal amounts of [³H]thymidine-labeled virions were allowed to attach to () or be internalized into (□) cells as described in Materials and Methods. Cells were then washed, and the number of viral particles bound per cell was determined. The data were obtained from two to four independent experiments performed in triplicate. (B) To analyze the level of Ad attachment, equal amounts of indicated Ad vectors at an MOI of 8,000 genomes per cell were mixed with 3.5 × 10⁵ 293 cells in 100 μl of adhesion buffer and then allowed to attach for 1 h on ice. Then virus-containing medium was removed; cells were washed twice with ice-cold PBS and resuspended in 100 μl of adhesion buffer. Then 100 μl of lysis buffer was added to the cells, and DNA was extracted as described earlier (61) (Virus attachment). To estimate the amount of Ad loaded per sample, the same volume of corresponding Ad as used in the attachment study was mixed with 3.5 × 10⁵ 293 cells in 100 μl of adhesion buffer. Immediately after mixing, 100 μl of lysis buffer was added to cells, and DNA was extracted (Virus load). After purification, DNA concentration was measured spectrophotometrically, and 1 μg of each sample was applied on the agarose gel. Shown are the ethidium bromide-stained 1% agarose gel before blotting, demonstrating that similar amounts of genomic DNA were loaded (top), and the result of Southern blot hybridization of the transferred DNA with a ³²P-labeled 8-kb HindIII fragment of the Ad5 genome, corresponding to the E2 region (bottom). The conditions of DNA transfer and hybridization were described earlier (61). M, molecular weight marker. Arrows indicate the Ad DNA (bottom) or mixture of Ad and cellular DNA (top).

FIG. 4

K_a values of long-shafted Ad variants with different knob domains determined on 293 cells. To analyze the affinity of binding of different knob domains to their receptors, Scatchard plots (59) were made for long-shafted Ad vectors possessing Ad5, Ad9, or Ad35 knob domains. The K_a was calculated based on the slopes of the lines using standard Microsoft Excel software. To ensure binding in equilibrium, different amounts of [³H]thymidine-labeled Ad particles ranging between 2,000 and 200,000 genomes per cell were incubated with 3 × 10⁵ 293 cells for 3 h on ice as described in reference . For each vector and for each viral concentration, virus attachment was performed in triplicate. The number of receptor sites was extrapolated from the intercept of the lines with the abscissa.

FIG. 5

Transduction of 293, K562, and Y79 cells with chimeric Ad. Cells were infected with different MOIs (viral genomes per cell) for 1 h at 37°C. Virus-containing medium was removed, and cells were incubated in growth medium for 24 h before the percentage of GFP-positive cells was determined by flow cytometry. All infections were done in triplicate in at least two independent settings.

FIG. 6

Effects of anti-CAR and anti-αv antibody on the transduction properties of Ad vectors. 293 cells (3.5 × 10⁵) were preincubated for 1 h at 37°C with anti-CAR (RmcB) or anti-αv integrin (L230) monoclonal antibodies and then incubated with indicated Ad vectors at an MOI of 20 genomes per cell (for Ad5L, Ad5S, Ad5/9L, Ad5/35L, and Ad5/35S) or 100 genomes per cell (Ad5/9S) for 1 h. Virus-containing medium was then removed, and the cells were incubated at 37°C for 24 h in growth medium before the percentage of GFP-positive cells was estimated by flow cytometry. As a control, cells were preincubated with an irrelevant anti-BrdU monoclonal antibody for 1 h before infection. A statistically significant decrease in the percentage of GFP-positive cells was found in infections with chimeric viruses, after preincubation of cells with anti-CAR (for Ad5L, Ad5S, Ad5/9L, and Ad5/9S) or anti-αv integrin (for Ad5L, Ad5S, Ad5/9L, Ad5/9S, and Ad5/35L) monoclonal antibodies (n = 6; P < 0.1). Notably, preincubation with the control antibody (Control) or with antibody dilution buffer only gave the same percentage of GFP-expressing cells (data not shown).

FIG. 7

Amino acid alignments of HVR1 in hexons from different Ad serotypes. Amino acid sequence alignments were done using the ClustalW 1.8 Global progressive algorithm at BCM Search Launcher (http://dot.imgen.bcm.tmc.edu:9331/multi-align). The different human Ad hexon protein sequences were obtained from the NCBI Protein Entrez data bank. Negatively charged amino acids are in bold, and positively charged residues are underlined. Stretches of positively charged amino acids in Ad8 and Ad9 hexon are boxed.

FIG. 8

Attachment of chimeric Ad vectors to 293 cells (A) and transduction of 293 cells with chimeric, CAR-binding Ad vectors (B and C) in the presence of Polybrene. (A) For attachment studies, 3.5 × 10⁵ 293 cells were incubated for 1 h on ice in 100 μl of adhesion buffer containing Polybrene (4 μg/ml). Then 25 μl of virus-containing medium was added to the cells (at a final MOI of 8,000 genomes per cell), and virus was allowed to attach for 1 h on ice. Cells were washed twice with PBS, and cell-associated radioactivity was measured n ≥ 3. (B) For transduction studies, 2.5 × 10⁵ 293 cells per well (12-well plate) were incubated with 400 μl of adhesion buffer containing Polybrene (4 μg/ml) at 37°C for 1 h. Then virus-containing medium (total volume of 100 μl) was added to cells. The final MOIs were 20 genomes per cell for Ad5L, Ad5S, and Ad5/9L and 100 genomes per cell for Ad5/9S. Virus was allowed to infect cells for 1 h at 37°C, and then the virus-containing medium was substituted with fresh medium; 24 h postinfection, cells were trypsinized, and the percentage of GFP-positive cells was determined by flow cytometry. (C) 293 cells infected in the presence of Polybrene with Ad vectors possessing modified fiber proteins 24 h postinfection. Cells were infected at MOIs and under the conditions described for panel B. Representative fields with comparable cell densities are shown for each variant. Magnification, ×400.

FIG. 9

Attachment of chimeric Ad vectors to 293 cells in the presence of Polybrene and anti-CAR monoclonal antibody. 293 cells (3.5 × 10⁵) were incubated in 100 μl of adhesion buffer without any competitors, with anti-CAR (RmcB monoclonal antibody; 1/200 dilution), with Polybrene (final concentration, 4 μg/ml), or with anti-CAR antibody and Polybrene together for 1 h on ice. Then Ad was added at an MOI of 4,000 genomes per cell in a total volume of 25 μl. Viruses were allowed to attach to cells for 1 h. After washing with ice-cold PBS, cells were pelleted, cell-associated radioactivity was measured, and the number of viral particles attached per cell was calculated for each virus. All attachment studies were performed in triplicate in at least two independent experiments.

FIG. 10

Attachment of chimeric Ad vectors to 293 cells in the presence of protamine (A) or Lipofectamine (B). Adsorption studies were performed as described for Fig. 9. The concentrations of protamine sulfate and the cationic lipid Lipofectamine were 400 and 50 μg/ml, respectively. All attachment studies were performed in triplicate in at least two independent experiments.