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 knobpossessing vectors, a long-shafted fiber was critical for efficient adsorption/internalization and transduction of CAR/␣v 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/␣v 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 ␣v integrin-mediated infections are influenced by other factors, including the length of the fiber shaft.Most recombinant adenovirus (Ad) vectors currently used for in vitro and in vivo gene transfer are based on serotype 5 (Ad5). Efficient transduction with these vectors requires the presence of appropriate cell receptors for binding and/or internalization. High-affinity binding of Ad5 is mediated through the coxsackievirus and Ad receptor (CAR) (6, 7, 68). Ad5 internalization requires an additional interaction of RGD motifs on the penton base with ␣v␤3 and/or ␣v␤5 integrins on the cell surface (32,39,48,71).Safety and efficacy of in vivo applications of Ad vectors require targeting to specific tissues, which is precluded by the widespread distribution of CAR and ␣v integrins. On the other hand, a number of tissues, which represent important targets for gene therapy, are refractory to Ad5 infection due to the lack of these receptors (11,64,70,73). Therefore, modification of tropism is one of the central tasks in Ad vector development. One strategy to retarget Ad5 vectors involves complexing the Ad capsid with bispecific antibodies (18, 70) or peptide ligands (17, 58). Another approach is genetic modification of the Ad5 fiber knob (43), which is aimed toward the incorporation of specific peptide ligands into the Ad5 knob with simultaneous abrogat...

Structural and Mutational Analysis of Human Ad37 and Canine Adenovirus 2 Fiber Heads in Complex with the D1 Domain of Coxsackie and Adenovirus Receptor

“…This may be because the HAd37 fiber shaft is too short and rigid to allow efficient binding to cellular CAR when the fiber is attached to the virus (38). Other studies also confirm that the length and flexibility of the fiber shaft is critical in infectivity (39).…”

Evaluation of Cell-Penetrating Peptide/Adenovirus Particles for Transduction of CAR-Negative Cells

“…The crucial role played by fiber and knob proteins of the virus in transduction prompts the genetic modification of these proteins. Studies have shown that transduction efficiency of the virus has shown dependency on the length of the fiber protein (Legrand, Spehner et al 1999;Shayakhmetov and Lieber 2000;Vigne, Dedieu et al 2003). Further, complete removal of the knob domain has been established as a way to 54 ablate tropism (Magnusson, Hong et al 2001).…”

Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo

“…Human reovirus T3D was kindly provided by Terence Dermody (Vanderbilt University, Nashville, TN). Viruses were banded in CsCl gradients, dialyzed, and stored in aliquots, as described (34). Adenovirus genome titers were determined by quantitative real-time PCR (for Ad5RFP and Ad5BAP) and OD260 measurement (for wildtype viruses).…”

“…An Ad5-FX complex was prepared by mixing 100 l of the Ad5S vector (34), which has a short-shafted fiber (1 ϫ 10 13 virus particles per ml) with 1 l of FX (2 mg/ml). Six-microliter aliquots of the Ad5-FX complex were applied to Quantifoil R2/4 holey carbon grids (Quantifoil Micro Tools) and a Vitrobot cryofixation device (FEI) was used for blotting and sample vitrification in liquid ethane.…”