The Ad5 fiber mediates nonviral gene transfer in the absence of the whole virus, utilizing a novel cell entry pathway

Abstract

The interesting discovery reported here that soluble adenovirus serotype 5 (Ad5) fiber proteins enter cells without the virus was a serendipitous result during our development of Ad5 capsid proteins as nonviral gene transfer vectors. The Ad5 capsid fiber and penton proteins mediate infection. The fiber docks to a noninternalizing cell surface protein called the coxsackievirus-Ad receptor (CAR), followed by penton binding to integrins, triggering integrin-mediated endocytosis of the virus. In our previous work, we assembled the nonviral complex, 3PO, which utilized the penton to mediate gene transfer through integrin binding and endocytosis. Here, we tested whether incorporating the fiber targets 3PO to CAR, thus recapitulating the Ad5 infection pathway. As CAR is not an endocytic receptor, we were surprised to find that the fiber alone, without the penton, enabled gene transfer by binding CAR, but internalizing through an unknown mechanism. We show here that the fiber distributes to the nucleus and cytoplasm after temperature-independent uptake, whereas the penton accumulates around the nucleus after temperature-dependent uptake. Fiber uptake by HeLa cells is also actin-dependent, requires the fiber tail/shaft region, and is largely inhibited by heparin. This study raises the possibility that alternative pathways may enable both viral and nonviral cell entry. Gene Therapy (2005) 12, 225-237.

Distinct Apical and Basolateral Membrane Requirements for Stretch-induced Membrane Traffic at the Apical Surface of Bladder Umbrella Cells

“…Membrane trafficking events are generally thought to be temperature sensitive, which reflects changes in protein folding and lipid fluidity at low temperatures as well as the presence of a thermodynamic barrier that is not conducive to membrane traffic. However, endocytosis of some viral proteins is only partially inhibited at 4°C, and exocytosis is observed in garter snake nerve terminals, the Xenopus pars intermedia, and the type II pneumocytes of hibernating squirrels at reduced temperatures (Elliot and O'Hare, 1997;Ormond et al, 2003;Tonosaki et al, 2004;Rentsendorj et al, 2005;Richard et al, 2005;Teng and Wilkinson, 2005). These studies indicate that exocytosis/endocytosis are not fundamentally impossible at reduced temperatures.…”

Intracellular trafficking of nonviral vectors

“…In agreement, we have observed large vesicles resembling macropinosomes during the temperature-independent, heparin-sensitive uptake of recombinant Ad5 fiber protein by HeLa cells. 117 Consistent with a macropinocytic route, fiber uptake and intracellular trafficking required intact actin. With regard to TAT-fusion uptake, membrane ruffling inhibitors amiloride and cytochalasin D blocked cell entry, as did cholesterol depletion using MbCD, and cholesterol sequestration using nystatin.…”

“…Transfection was blocked by pretreatment with sulfated polysaccharides, fucoidan, heparin, or dextran sulfate. These studies were expanded in vivo 51 and enter these cells through a heparin-inhibited pathway, implicating heparan sulfate glycosaminoglycans as contributing to binding. Moreover, complexes known as 'dodecahedrons', assembled from Ad3 penton base proteins, also bind and enter CHO cells by proteoglycan interaction.…”

“…We have found that the Ad5 fiber protein exhibited an unexpected ability to facilitate gene transfer of protamine-DNA complexes. 51 Transfection by the fibermediated complex, called FPO, required binding to the Ad5 receptor, CAR, but the receptor itself did not endocytose. As the fiber is not known to be the cell entry protein for Ad5 infection, its mode of uptake and trafficking without the virus was examined.…”

“…Some recent studies in secretory epithelial cells of the lacrimal (tear) gland suggest that the ability of molecular conjugates to 'hijack' intracellular trafficking pathways in order to rapidly target the nucleus is dependent upon the cell type. For example, a recombinant Ad5 penton base protein, which has been incorporated into several types of molecular conjugates for its endosomolytic and intracellular trafficking capacity, efficiently internalized into intracellular sites in HeLa cells, 51,187 but was observed at the surface of primary acinar epithelial cells even after 24 h of sustained incubation, with no evidence for penton internalization or recovery in cytoplasm or intracellular organelles. 188 In contrast, Ad5 efficiently entered these cells in a pattern consistent with endocytosis, 188 following the same general profile as shown in other systems.…”