This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parker, A. L.
Right arrow Articles by Baker, A. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parker, A. L.
Right arrow Articles by Baker, A. H.

 Previous Article  |  Next Article 

Journal of Virology, April 2007, p. 3627-3631, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.02786-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Influence of Coagulation Factor Zymogens on the Infectivity of Adenoviruses Pseudotyped with Fibers from Subgroup D{triangledown}

Alan L. Parker,1 John H. McVey,2 Jessica H. Doctor,1 Oscar Lopez-Franco,1 Simon N. Waddington,3 Menzo J. E. Havenga,4 Stuart A. Nicklin,1 and Andrew H. Baker1*

British Heart Foundation Glasgow Cardiovascular Research Centre, Division of Cardiovascular and Medical Sciences, University of Glasgow, 126 University Place, Glasgow G12 8TA, United Kingdom,1 Haemostasis and Thrombosis, MRC Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom,2 Department of Haematology, Haemophilia Centre and Haemostasis Unit, Royal Free and University College Medical School, London NW3 2PF, United Kingdom,3 Crucell, P.O. Box 2048, 2301 CA Leiden, The Netherlands4

Received 18 December 2006/ Accepted 16 January 2007


arrow
ABSTRACT
 
Recent evidence supports a role for vitamin K-dependent coagulation zymogens in adenovirus serotype 5 (Ad5, subgroup C) infection of hepatocytes. Here, we assessed the effect of virus-zymogen interaction on cellular transduction using a panel of fiber (f)-pseudotyped viruses derived from subgroup D (f47, f33, f24, f45, f17, f30). Each virus directly bound factor X (FX) as determined by surface plasmon resonance, resulting in enhanced cell surface binding. Infection of HepG2 cells was promoted by FX but not by FVII or FIX, while transduction of CHO cells was blocked in heparan sulfate proteoglycan-deficient cells. This suggests a broad role for FX in adenovirus infectivity.


arrow
TEXT
 
Adenoviral vectors, particularly adenovirus serotype 5 (Ad5, subgroup C virus), are used routinely for gene therapy in the experimental setting and are being tested extensively in the clinical setting. In vitro, Ad5 binds to cells predominantly through primary interaction with the coxsackie virus and adenovirus receptor (3, 18) and internalization through integrin engagement (19). The profound in vivo infectivity of Ad5 for the liver following systemic delivery, however, is dictated by the virus binding to coagulation zymogens, including factor IX (FIX) (16) and FX (15). This is particularly relevant for understanding adenovirus infectivity following intravascular administration, since the virus comes into immediate contact with blood cells (14) and circulating proteins. For efficient retargeting of Ad5-based gene delivery vectors to alternate sites (e.g.,disseminated cancers, alternate organs, and specific sites of disease that lack medically achievable surgical access), it is likely that modulation of this pathway will be required, since binding of coagulation factors to the virus is efficient (15). Previous studies have suggested that the fiber is the major determinant of FIX binding to the capsid (16). A simple method, therefore, to alter susceptibility to coagulation factor binding may be to use fibers derived from alternate serotypes and pseudotyped onto the Ad5 capsid. Shayakhmetov and coworkers demonstrated that Ad5 capsids possessing the fiber of serotype 35 (subgroup B) showed enhancement of cell infection mediated by FIX (16). Subgroup D-based Ads, either as pseudotyped viruses or as complete serotype viruses, are being developed for a variety of clinical applications, including targeted in vivo gene delivery and vaccination (5, 12). Additional virological importance may be assigned to coagulation factor binding, since adenovirus infections are relatively common (and heterogeneous with respect to adenovirus subgroups, including subgroup D) in immunocompromised patients (13). Many of the receptors for adenoviruses from this subgroup remain to be isolated, and those that have been characterized often bind with relatively low affinity (1, 2, 4, 5). Since virus exposure to the blood and hence dissemination may occur in a number of such applications, it is important to assess coagulation factor binding capacity. We documented this capacity by using a panel of luciferase-expressing Ad5 vectors pseudotyped with fibers from subgroup D, including f17, f24, f30, f33, f45, and f47 (see Fig. 1A for a phylogenetic depiction) (7-9).


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		FIG. 1. Subgroup D viruses and surface plasmon resonance analysis of FX-virus binding. (A) Phylogenetic depiction of subgroup D viruses, adapted from previous work (9). (B) Adenovirus Ad5/f45 was perfused over FX, and FXI was immobilized onto a CM5 sensor chip in 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.005% Tween 20 at a flow rate of 20 µl/m at 25°C. Shown are typical sensorgrams indicating an association with FX but not FXI and an undetectable dissociation of the virus from FX following the end of the injection but a ready dissociation upon injection of 3 mM EDTA. RU, response units.

We first screened viruses for coagulation factor binding by using surface plasmon resonance. We used biosensor chips with immobilized FX or FXI (negative control) (15). All viruses bound to FX but not to FXI (see Fig. 1B for Ad5/f45 as an example). In each case, and consistent with previous data for Ad5 (15), no dissociation of virus from FX was observed until 3 mM EDTA was added, indicating a strong and calcium-dependent interaction with FX (see Fig. 1B for Ad5/f45 as an example). This demonstrates that all viruses evaluated directly bind to FX.

We next sought to determine whether physiological concentrations of FX could influence HepG2 cell binding and transduction mediated by each pseudotyped adenovirus in vitro. Previously we have used this in vitro model as a reproducible model system to examine the effect of coagulation factors on human cell transduction (15). We exposed cells and viruses to physiological concentrations of FX (10 µg/ml, 1 IU/ml) in serum-free medium for 1 h at 4°C (for cell binding studies) or for 3 h at 37°C followed by incubation for 72 h (for transduction studies). All subgroup D fiber-pseudotyped vectors showed a significant increase in cell binding (Fig. 2A) and subsequent transgene expression (Fig. 2B) in HepG2 cells in the presence of FX, ranging from a 3.7-fold increase in transduction in the presence of FX for serotype Ad5/f30 to a 24.9-fold increase for Ad5/f33 (Fig. 2B). This demonstrates that physiological FX concentrations substantially enhance cell binding and transduction of HepG2 cells mediated by each subgroup D fiber-pseudotyped virus.


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 2. Effect of physiological FX concentrations on Ad-mediated binding and transduction of HepG2 cells. HepG2 cells were infected with each Ad (1,000 viral particles/cell) in the absence or presence of physiological levels of FX for various times in serum-free medium. (A) Cell binding analysis by TaqMan of recovered viral genomes following exposure to cells at 4°C for 1 h. Representative TaqMan traces are shown for Ad5/f17 and Ad5/f47 in the presence and absence of FX. u.i., uninfected cells; {Delta}Rn, change in normalized fluorescence. (B) Cell transduction by luciferase and total protein quantification at 72 h postinfection following exposure to FX for 3 h at 37°C. *, P < 0.05 versus virus in the absence of FX; #, P < 0.01 versus virus in the absence of FX (Student's t test). The numbers above the bars represent the changes (n-fold) in comparison with the value in the absence of FX. This figure is representative of three independent experiments. RLU, relative light units.

To further investigate the effect of other coagulation factors with the {gamma}-carboxyglutamic acid-epidermal growth factor-like (EGF)-EGF-serine protease domain structure on virus transduction, we transduced HepG2 cells with a subset of viruses, Ad5/f47, Ad5/f33, and Ad5/f30, in the presence of physiological concentrations of FVII, FIX, FX, or FXI (negative control) (Fig. 3). All reagents were purified proteins obtained from Hematological Technologies, Ltd., except FVII, which was recombinant (15). All subgroup D-pseudotyped viruses showed a substantial enhancement of HepG2 cell transduction in the presence of FX but not with FVII, FIX, or FXI (Fig. 3). The lack of effect of FVII and FIX was surprising based on previous observations for Ad5 (15, 16) and for the subgroup B Ad35 (16). Although further experiments may be required to assess direct interactions of individual coagulation factors and each virus as well as recombinant forms of FIX, these data do suggest potential subgroup-specific differences in fiber-coagulation factor interactions.


Figure 3
View larger version (8K):
[in this window]
[in a new window]

  		FIG. 3. Effects of FVII, FIX, and FX on transduction of HepG2 cells. HepG2 cells were incubated with 1 IU/ml of human FVII, FIX, or FX or the divergent control zymogen FXI in the absence or presence of each Ad (1,000 viral particles/cell) for 3 h. Transgene expression was quantified at 72 h postinfection and normalized to the total protein content. *, P < 0.05 versus virus in the absence of FX; #, P < 0.01 versus virus in the absence of FX (Student's t test). This figure is representative of three independent experiments. RLU, relative light units.

We hypothesized that the increase in transduction mediated through FX would be via an interaction with heparan sulfate proteoglycan (HSPG), as observed for Ad5 (15, 16); therefore, CHO cell infections were performed for each virus. CHO-K1 cells (wild type) express HSPGs, whereas CHO-pgsA745 cells have been genetically modified to be deficient in polymerization of HSPGs and therefore have no cell surface expression (6). In each case, physiological levels of FX enhanced transduction in CHO-K1 cells (Fig. 4). However, this level was significantly reduced for CHO-pgsA745 cells (Fig. 4). Therefore, for subgroup D fiber-pseudotyped viruses that show an enhancement in cell binding and transduction through FX, the virus is targeted through HSPG, consistent with the mechanism of retargeting of Ad5 (15, 16). It is noteworthy that the putative HSPG binding site in the third pseudorepeat of the Ad5 fiber (17) is not present in any of the pseudotypes here. However, we recently demonstrated that Ad5 fibers mutated at this site (KKTK motif) show efficient binding to FX (11). Taken together, these data suggest that Ad5 and subgroup D fiber-pseudotyped Ads that are responsive to FX mediate enhanced cell transduction through retargeting to HSPG on the cell surface. This suggests that coagulation factors play a broad role in dictating adenovirus tropism when exposure to blood occurs, since subgroup C- (15, 16), subgroup B- (16), and now subgroup D-based viruses are all sensitive to FX binding. This is in contrast to a recent finding that Ad5 (and other subgroup C viruses) but not representative viruses from all other subgroups used lactoferrin as a bridge for coxsackie virus and adenovirus receptor-independent transduction of A549 cells (10). This demonstrates that research into coagulation factors and other proteins that can target the Ad capsid proteins in vivo (either following localized exposure in the case of tear fluid and lactoferrin [10] or via exposure to the bloodstream) is warranted, since this has fundamental implications for receptor utilization and infectivity in vivo.


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 4. Effect of FX on virus transduction of CHO-K1 and CHO-pgsA745 cells. CHO-K1 and CHO-pgsA745 cells were infected with 5,000 viral particles/cell of each Ad in the absence or presence of 1 IU/ml FX for 3 h at 37°C, the cells were washed, and the number of transgenes was quantified at 72 h postinfection and normalized to the total protein content. *, P < 0.05 versus virus in the absence of FX (Student's t test). This figure is representative of three independent experiments. RLU, relative light units.


arrow
ACKNOWLEDGMENTS
 
This work was supported by the European Commission and Biotechnology and Biophysical Research Council for funding support. The Haemostasis and Thrombosis department is supported by the Medical Research Council. S.N.W. is a Philip Gray Memorial Fellow, Katharine Dormandy Trust.

We thank Nicola Britton and Gregor Aitchison for technical assistance.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: British Heart Foundation Glasgow Cardiovascular Research Centre, Division of Cardiovascular and Medical Sciences, University of Glasgow, 126 University Place, Glasgow G12 8TA, United Kingdom. Phone: 44 141 330 1977. Fax: 44 141 330 6997. E-mail: ab11f{at}clinmed.gla.ac.uk. Back

{triangledown} Published ahead of print on 24 January 2007. Back


arrow
REFERENCES
 
    1
  1. Arnberg, N., A. H. Kidd, K. Edlund, F. Olfat, and G. Wadell. 2000. Initial interactions of subgenus D adenoviruses with A549 cellular receptors: sialic acid versus {alpha}v integrins. J. Virol. 74:7691-7693.[Abstract/Free Full Text]
    2. 2
  2. Arnberg, N., P. Pring-Åkerblom, and G. Wadell. 2002. Adenovirus type 37 uses sialic acid as a cellular receptor on Chang C cells. J. Virol. 76:8834-8841.[Abstract/Free Full Text]
    3. 3
  3. Bergelson, J. M., J. A. Cunningham, G. Droguett, E. A. Kurt-Jones, A. Krithivas, J. S. Hong, M. S. Horwitz, R. L. Crowell, and R. W. Finberg. 1997. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320-1323.[Abstract/Free Full Text]
    4. 4
  4. Burmeister, W. P., D. Guilligay, S. Cusack, G. Wadell, and N. Arnberg. 2004. Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites. J. Virol. 78:7727-7736.[Abstract/Free Full Text]
    5. 5
  5. Denby, L., L. Work, D. Graham, C.-L. Hsu, D. von Seggern, N. S. A., and A. Baker. 2004. Adenoviral serotype 5 vectors pseudotyped with fibers from subgroup D show modified tropism in vitro and in vivo. Hum. Gene Ther. 15:1054-1064.[Medline]
    6. 6
  6. Esko, J. D., T. E. Stewart, and W. H. Taylor. 1985. Animal cell mutants defective in glycosaminoglycan biosynthesis. Proc. Natl. Acad. Sci. USA 82:3197-3201.[Abstract/Free Full Text]
    7. 7
  7. Haberberger, T. C., K. Kupfer, and J. E. Murphy. 2000. Profiling of genes which are differentially expressed in mouse liver in response to adenoviral vectors and delivered genes. Gene Ther. 7:903-909.[CrossRef][Medline]
    8. 8
  8. Havenga, M. J. E., A. A. C. Lemckert, J. M. Grimbergen, R. Vogels, L. G. M. Huisman, D. Valerio, A. Bout, and P. H. A. Quax. 2001. Improved adenovirus vectors for infection of cardiovascular tissues. J. Virol. 75:3335-3342.[Abstract/Free Full Text]
    9. 9
  9. Havenga, M. J. E., A. A. C. Lemckert, O. J. A. E. Ophorst, M. van Meijer, W. T. V. Germeraad, J. Grimbergen, M. A. van den Doel, R. Vogels, J. van Duetekom, A. A. M. Janson, J. D. de Bruijn, F. Uytdehaag, P. H. A. Quax, T. Logtenberg, M. Mehtali, and A. Bout. 2002. Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease. J. Virol. 76:4612-4620.[Abstract/Free Full Text]
    10. 10
  10. Johansson, C., M. Jonsson, M. Marttila, D. Persson, X.-L. Fan, J. Skog, L. Frängsmyr, G. Wadell, and N. Arnberg. 2007. Adenoviruses use lactoferrin as a bridge for CAR-independent binding to and infection of epithelial cells. J. Virol. 81:954-963.[Abstract/Free Full Text]
    11. 11
  11. Kritz, A. B., C. G. Nicol, K. L. Dishart, R. Nelson, S. Holbeck, D. J. Von Seggern, L. M. Work, J. H. McVey, S. A. Nicklin, and A. H. Baker. 23 January 2007. Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop. Mol. Ther. doi:10.1038/sj.mt.6300094.
    12. 12
  12. Lecollinet, S., F. Gavard, M. J. E. Havenga, O. B. Spiller, A. Lemckert, J. Goudsmit, M. Eloit, and J. Richardson. 2006. Improved gene delivery to intestinal mucosa by adenoviral vectors bearing subgroup B and D fibers. J. Virol. 80:2747-2759.[Abstract/Free Full Text]
    13. 13
  13. Lion, T., R. Baumgartinger, F. Watzinger, S. Matthes-Martin, M. Suda, S. Preuner, B. Futterknecht, A. Lawitschka, C. Peters, U. Potschger, and H. Gadner. 2003. Molecular monitoring of adenovirus in peripheral blood after allogeneic bone marrow transplantation permits early diagnosis of disseminated disease. Blood 102:1114-1120.[Abstract/Free Full Text]
    14. 14
  14. Lyons, M., D. Onion, N. K. Green, K. Aslan, R. Rajaratnam, M. Bazan-Peregrino, S. Phipps, S. Hale, V. Mautner, L. W. Seymour, and K. D. Fisher. 2006. Adenovirus type 5 interactions with human blood cells may compromise systemic delivery. Mol. Ther. 14:118-128.[CrossRef][Medline]
    15. 15
  15. Parker, A. L., S. N. Waddington, C. G. Nicol, D. M. Shayakhmetov, S. M. Buckley, L. Denby, G. Kemball-Cook, S. Ni, A. Lieber, J. H. McVey, S. A. Nicklin, and A. H. Baker. 2006. Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes. Blood 108:2554-2561.[Abstract/Free Full Text]
    16. 16
  16. Shayakhmetov, D. M., A. Gaggar, S. Ni, Z.-Y. Li, and A. Lieber. 2005. Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity. J. Virol. 79:7478-7491.[Abstract/Free Full Text]
    17. 17
  17. Smith, T., N. Idamakanti, M. Rollence, J. Marshall-neff, J. Kim, K. Mulgrew, G. Nemerow, M. Kaleko, and S. Stevenson. 2003. Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice. Hum. Gene Ther. 14:777-787.[CrossRef][Medline]
    18. 18
  18. Tomko, R. P., R. Xu, and L. Philipson. 1997. HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc. Natl. Acad. Sci. USA 94:3352-3356.[Abstract/Free Full Text]
    19. 19
  19. Wickham, T. J., P. Mathias, D. A. Cheresh, and G. R. Nemerow. 1993. Integrins {alpha}vß3 and {alpha}vß5 promote adenovirus internalization but not virus attachment. Cell 73:309-319.[CrossRef][Medline]

Journal of Virology, April 2007, p. 3627-3631, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.02786-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jonsson, M. I., Lenman, A. E., Frangsmyr, L., Nyberg, C., Abdullahi, M., Arnberg, N. (2009). Coagulation Factors IX and X Enhance Binding and Infection of Adenovirus Types 5 and 31 in Human Epithelial Cells. J. Virol. 83: 3816-3825 [Abstract] [Full Text]  
  • Raman, S., Hsu, T.-H., Ashley, S. L., Spindler, K. R. (2009). Usage of Integrin and Heparan Sulfate as Receptors for Mouse Adenovirus Type 1. J. Virol. 83: 2831-2838 [Abstract] [Full Text]  
  • Parker, A. L., Waddington, S. N., Buckley, S. M. K., Custers, J., Havenga, M. J. E., van Rooijen, N., Goudsmit, J., McVey, J. H., Nicklin, S. A., Baker, A. H. (2009). Effect of Neutralizing Sera on Factor X-Mediated Adenovirus Serotype 5 Gene Transfer. J. Virol. 83: 479-483 [Abstract] [Full Text]  
  • Waddington, S. N., Parker, A. L., Havenga, M., Nicklin, S. A., Buckley, S. M. K., McVey, J. H., Baker, A. H. (2007). Targeting of Adenovirus Serotype 5 (Ad5) and 5/47 Pseudotyped Vectors In Vivo: Fundamental Involvement of Coagulation Factors and Redundancy of CAR Binding by Ad5. J. Virol. 81: 9568-9571 [Abstract] [Full Text]  
  • Stone, D., Liu, Y., Shayakhmetov, D., Li, Z.-Y., Ni, S., Lieber, A. (2007). Adenovirus-Platelet Interaction in Blood Causes Virus Sequestration to the Reticuloendothelial System of the Liver. J. Virol. 81: 4866-4871 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parker, A. L.
Right arrow Articles by Baker, A. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parker, A. L.
Right arrow Articles by Baker, A. H.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»