Identification of Contact Residues and Definition of the CAR-Binding Site of Adenovirus Type 5 Fiber Protein

Kirby, Ian; Davison, Elizabeth; Beavil, Andrew J.; Soh, Cecilia; Wickham, Thomas J.; Roelvink, Peter W.; Kovesdi, Imre; Sutton, Brian J.; Santis, George

doi:10.1128/jvi.74.6.2804-2813.2000

Cited by 155 publications

(144 citation statements)

References 31 publications

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“…Another group also confirmed, by surface plasmon resonance and Gene Therapy cell-binding competition experiments with recombinant mutated Ad fibers, that residues located in the AB and the DE loops and in ␤-strand E and F are directly or indirectly in contact with the cellular receptor. 20,21 In the present study, fiber residues involved in CAR binding were identified. We have studied the impact of mutations on receptor binding in a viral context and on the biochemical properties of the corresponding viruses.…”

Section: Introductionmentioning

confidence: 75%

“…Recently, structural analysis and site-directed mutagenesis have led to the identification of key determinants of binding specificity. [19][20][21] In addition, Roelvink et al 18 demonstrated the feasibility of simultaneously ablating the CAR-binding site of the Ad knob and redirecting the virus to a new receptor in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…The mutations are located in the AB loop (Ser408Glu) and in the DG loop (Tyr491Asp, Ala494Asp, Phe497Asp, Met498Asp, Pro499-Gly, Ala503Asp), consistent with the results recently obtained by others. 18,19,21 In addition, the modified Leu48-5Asp-fiber virus still efficiently infects cells through CAR binding ( Figure 1). Interestingly, the substitution of the same residue to a lysine (Leu485Lys) has been shown to abolish high-affinity binding of the modified protein to CAR.…”

Section: Generation Of Fiber-modified Adenovirusesmentioning

confidence: 99%

“…Interestingly, the substitution of the same residue to a lysine (Leu485Lys) has been shown to abolish high-affinity binding of the modified protein to CAR. 21 This result can be explained by the observation that Leu485 of Ad5 is structurally equivalent to Ser497 of Ad12, which in the Ad12 knob-CAR context is in close proximity to a positively charged region of CAR. Therefore, it appears logical that introducing a positively charged lysine at position 485 abrogates CAR-binding, while an aspartate has less or no effect.…”

Section: Generation Of Fiber-modified Adenovirusesmentioning

confidence: 99%

“…15,16 Only recently, through sequence analysis and mutagenesis of the knob domain of the Ad5, has a conserved receptor-binding region been identified. [17][18][19][20][21] These authors demonstrated that the CARbinding site consists of residues from the AB loop, the B ␤-sheet and the DE loop, while the FG loop seems to play an indirect role in binding CAR. As this site is located on the side of the knob monomer, these findings imply that the trimeric knob could bind to three CAR molecules independently.…”

Section: Introductionmentioning

confidence: 99%

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Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

et al. 2001

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Targeting of adenovirus (Ad)-encoded therapeutic genes to specific cell types has become a major goal in gene therapy. Redirecting the specificity of infection requires the abrogation of the natural interaction between the viral fiber and its cellular receptors (CAR) and the simultaneous introduction of a new binding specificity into the viral capsid. To abrogate the natural affinity of the fiber, we have mutated residues presumed to be directly or indirectly involved in CARbinding in the knob domain of the fiber protein. These residues are located in the AB loop (Ser408) and in the DG loop (Tyr491, Ala494, Ala503). The mutations Ser408Glu,

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Section: Introductionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Generation Of Fiber-modified Adenovirusesmentioning

confidence: 99%

Section: Generation Of Fiber-modified Adenovirusesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

et al. 2001

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Gene Therapy: Introduction and Overview

White¹,

Samulski²

2005

Textbook of Hemophilia

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Retargeting of adenoviral infection to melanoma: Combining genetic ablation of native tropism with a recombinant bispecific single‐chain diabody (scDb) adapter that binds to fiber knob and HMWMAA

Nettelbeck

Rivera

Kupsch

et al. 2003

Intl Journal of Cancer

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Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50-fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAAmediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wildtype fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold increased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma.

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Identification of Contact Residues and Definition of the CAR-Binding Site of Adenovirus Type 5 Fiber Protein

Cited by 155 publications

References 31 publications

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

Gene Therapy: Introduction and Overview

Retargeting of adenoviral infection to melanoma: Combining genetic ablation of native tropism with a recombinant bispecific single‐chain diabody (scDb) adapter that binds to fiber knob and HMWMAA

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