Requirements for Receptor Engagement during Infection by Adenovirus Complexed with Blood Coagulation Factor X

Abstract

Human adenoviruses from multiple species bind to coagulation factor X (FX), yet the importance of this interaction in adenovirus dissemination is unknown. Upon contact with blood, vectors based on adenovirus serotype 5 (Ad5) binds to FX via the hexon protein with nanomolar affinity, leading to selective uptake of the complex into the liver and spleen. The Ad5:FX complex putatively targets heparan sulfate proteoglycans (HSPGs). The aim of this study was to elucidate the specific requirements for Ad5:FX-mediated cellular uptake in this high-affinity pathway, specifically the HSPG receptor requirements as well as the role of penton base-mediated integrin engagement in subsequent internalisation. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins/heparan sulfates significantly decreased FX-mediated Ad5 cell binding in vitro and ex vivo. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 cellular uptake. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration of adenovirus. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or α _v integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface involves HSPG sulfation. Interestingly, Ad5 vectors ablated for α _v integrin binding showed markedly delayed cell entry, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and transport following surface binding mediated through FX. This study therefore integrates the established model of α _v integrin-dependent adenoviral infection with the high-affinity FX-mediated pathway. This has important implications for mechanisms that define organ targeting following contact of human adenoviruses with blood.

Author Summary

Adenoviruses can infect many cell types and cause a range of illnesses in humans, including respiratory, ocular and gastrointestinal disorders. These illnesses are rarely fatal; however, in immunocompromised individuals, especially young children, disseminated adenovirus infections can cause serious and life-threatening complications. Studies have shown that several adenoviruses including vectors based on adenovirus serotype 5 (Ad5) bind to coagulation factor X (FX) in the bloodstream. Ad5 uses the high-affinity interaction with FX to putatively bind to heparan sulfate proteoglycans (HSPGs). However, very little is known about this infection pathway. Here we demonstrate that interaction of Ad5:FX with HSPGs is solely via the HS sidechains of these ubiquitously-expressed molecules. We further show that this interaction is dependent on HS sulfation, in particular O-sulfation. Although attachment of Ad5:FX to HSPGs is independent of the coxsackievirus and adenovirus receptor (CAR) or α _v integrins, efficient and rapid intracellular transport of Ad5 retains a dependence on engagement of α _v integrins via the penton base protein. This is the first study to characterise the receptor requirements for cell uptake via the recently-identified, FX-mediated infection pathway, which may be of significance for the development of therapies against disseminated adenoviral disease.