Adenovirus Fiber Shaft Contains a Trimerization Element That Supports Peptide Fusion for Targeted Gene Delivery

Abstract

Adenoviral (Ad) vectors have been widely used in human gene therapy clinical trials. However, their application has frequently been restricted by the unfavorable expression of cell surface receptors critical for Ad infection. Infections by Ad2 and Ad5 are largely regulated by the elongated fiber protein that mediates its attachment to a cell surface receptor, coxsackie and adenovirus receptor (CAR). The fiber protein is a homotrimer consisting of an N-terminal tail, a long shaft, and a C-terminal knob region that is responsible for high-affinity receptor binding and Ad tropism. Consequently, the modification of the knob region, including peptide insertion and C-terminal fusion of ligands for cell surface receptors, has become a major research focus for targeting gene delivery. Such manipulation tends to disrupt fiber assembly since the knob region contains a stabilization element for fiber trimerization. We report here the identification of a novel trimerization element in the Ad fiber shaft. We demonstrate that fiber fragments containing the N-terminal tail and shaft repeats formed stable trimers that assembled onto Ad virions independently of the knob region. This fiber shaft trimerization element (FSTE) exhibited a capacity to support peptide fusion. We showed that Ad, modified with a chimeric protein by direct fusion of the FSTE with a growth factor ligand or a single-chain antibody, delivered a reporter gene selectively. Together, these results indicate that the shaft region of Ad fiber protein contains a trimerization element that allows ligand fusion, which potentially broadens the basis for Ad vector development.

Adenoviruses: update on structure and function

“…A further~180 aa form the C-terminal globular head or knob. Trimerization is governed by sequences in both the knob and shaft regions (Li et al, 2006). The knob contains eight-stranded b-barrels in each subunit and has a central depression with three symmetry-related valleys.…”

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

“…Recently, an additional endogenous trimerization element was identified (although its precise sequence was not elucidated) within the N-terminus of the fiber shaft [ 194 ]. In this study, the authors’ generated truncated fiber constructs corresponding to the N-terminal tail with the first 6.5, 7, 7.5 and 9 shaft repeats, all of which were found to retain the ability to form stable homotrimeric fibers independently of the C-terminal knob.…”

Construction of Metabolically Biotinylated Adenovirus with Deleted Fiber Knob as Targeting Vector

“…Without the knob domain, a shortened fiber protein 9R containing the N-terminal tail and a shaft with 9 repeating sequences can form stable trimers and support peptide fusion [10]. Also a truncated form of PSTCD fused to the C-terminus of the fiber protein can be efficiently biotinylated by human holocarboxylase synthetase presented in HEK-293 cells [11].…”

“…The hAd5 fiber proteins exist as homotrimers which contains an N-terminal tail, a central shaft comprising 21 repeating sequences of 15 amino acids, and a C-terminal globular knob domain [ 9 ]. Without the knob domain, a shortened fiber protein 9R containing the N-terminal tail and a shaft with 9 repeating sequences can form stable trimers and support peptide fusion [ 10 ]. Also a truncated form of PSTCD fused to the C-terminus of the fiber protein can be efficiently biotinylated by human holocarboxylase synthetase presented in HEK-293 cells [ 11 ].…”