Abstract
Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon∶FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.
Highlights
The human adenovirus (HAdV) capsid is composed of eleven well identified structural proteins, of which the hexon is the major component with 240 copies forming the 20 facets and 30 edges of the icosahedral capsid
gamma-carboxylic acid (Gla) domain-dependence of factor X (FX)-mediated binding of serotype 5 hexon protein and adenovirions to heparan sulfate in vitro The interaction between soluble HAdV5wt hexon protein and heparan sulfate in vitro, directly or indirectly via FX, was investigated using surface plasmon resonance (SPR) analysis and a hypersulfated form of heparin (HS), recognized as the best structural model to mimic the heparan sulfate chains contained in heparan sulfate proteoglycan (HSPG) [48,49]
As expected from previous studies, we found that hexon binding to HS was enhanced in the presence of FX, but not with FXGL (Fig. 1 A), and this enhancing effect occurred in a FX dose-dependent manner
Summary
The human adenovirus (HAdV) capsid is composed of eleven well identified structural proteins, of which the hexon is the major component with 240 copies forming the 20 facets and 30 edges of the icosahedral capsid. The classical cell entry and trafficking pathway of HAdV5, as demonstrated by epithelial cell models of adenoviral infection in vitro, consists of (i) the fiber binding to CAR, the Coxsackie B and Adenovirus Receptor [4,5,6,7,8,9,10,11], followed by (ii) the interaction of the penton base RGD motifs with the cellular integrins alphaVbeta and alphaVbeta5, [12,13,14,15], which promotes virus endocytosis into clathrin-coated vesicles [16,17]. In step (iii), partially uncoated HAdV5 particles are released from the early endosomal compartment into the cytosol, a process involving the capsid protein VI [18,19]. (iv) Dynein and microtubules mediate the cytoplasmic transit of the residual HAdV5 capsid, which docks at the nuclear pore complex [20,21], before (v) the nuclear import of the viral nucleoprotein core [22,23]
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