Cell Entry and Trafficking of Human Adenovirus Bound to Blood Factor X Is Determined by the Fiber Serotype and Not Hexon:Heparan Sulfate Interaction

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.

A myeloid cell-binding adenovirus efficiently targets gene transfer to the lung and escapes liver tropism

“…This is an interesting finding, since previous studies have implicated both the fiber knob domain and hexon protein as important components for KC sequestration. 13, 38, 39 While our data do not directly demonstrate a mitigation of KC interaction with knob-deleted Ad particles, our study suggests that KC sequestration, and the acute immune response associated with KC uptake (as measured by serum IL-6 levls), 5, 6, 10 can be minimized through more efficient binding of Ad to other tissues, such as the lung. Thus, liver detargeting via additional capsid modifications may not be necessary, as long as the route of injection and the efficiency of targeting or sequestration supersede viral access to the liver.…”

Hexon Hypervariable Region-Modified Adenovirus Type 5 (Ad5) Vectors Display Reduced Hepatotoxicity but Induce T Lymphocyte Phenotypes Similar to Ad5 Vectors

“…Ad5 vectors at high doses also exhibit hepatotoxicity as a result of liver tropism due to the binding of blood coagulation factor X (FX) in the systemic circulation (7)(8)(9)(10). Both of these properties appear to be mediated primarily by the hexon hypervariable regions (HVRs) (8,(11)(12)(13)(14)(15)(16). HVRs are highly variable among Ad serotypes and represent the primary determinant of neutralization specificity (17)(18)(19)(20).…”

Microparticle-Mediated Transfer of the Viral Receptors CAR and CD46, and the CFTR Channel in a CHO Cell Model Confers New Functions to Target Cells

“…With MP 30 CHO, or at low doses of MP 30 CAR and MP 30 CD46 (≤5 MP 30 /cell), 2 to 10% cells were found to become GFP-positive ( Fig. 3 ), which corresponded to the background of infection of CHO cells by HAdV5 and HAdV5F35 via other cell surface molecules, such as heparan sulfate glucosaminoglycans, which act as alternative receptors for HAdV5 and HAdV5F35 [43], [64]. However, CHO cells pre-incubated with MP 30 CAR or MP 30 CD46, and infected with HAdV5-GFP and HAdV5F35-GFP, respectively, showed a 2- to 3-fold increase in GFP-positive cells.…”