Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer

Abstract

The adenovirus (Ad) penton base protein facilitates viral infection by binding cell surface integrins, triggering receptormediated endocytosis and mediating endosomal penetration. Given these multiple functions, recombinant penton base proteins have been utilized as non-viral vehicles for gene transfer by our lab and others. Although we have previously demonstrated that penton base-derived vectors undergo integrin-specific binding and cell entry, less than desirable levels of gene expression have led us to re-evaluate the recombinant penton base as an agent for gene delivery. To do so, we have examined here the intracellular trafficking of an Ad serotype 5 (Ad5) recombinant penton base protein (PB). Here, we not only observed that PB utilizes a similar, typical trafficking pathway of whole Ad, but also found that PB entered HeLa cells through pathways not yet identified as contributing to cell entry by the whole virus. We show by high-resolution confocal microscopy and biochemical methods that binding to a v -integrins is a requirement for cell entry, but that early internalization stages did not substantially pass through clathrin-positive and early endosomal compartments. Moreover, a subpopulation of internalized protein localized with caveolin-positive compartments and Golgi markers, suggesting that a certain percentage of proteins pass through nonclathrin-mediated pathways. Similar to the virus, trafficking toward the nucleus was affected by disruption of microtubules and dynein. The majority of penton base molecules avoided the lysosome while facilitating early vesicle release of low molecular weight dextran molecules. In further support of a vesicle escape capacity, a subpopulation of internalized penton base appeared to enter the nucleus, as observed by high-resolution confocal microscopy and cell fractionation. As a confirmation of these findings, we demonstrate that a recombinant penton base facilitated cytosolic entry of an siRNA molecule as observed by RNA interference of a marker gene. Based on our findings here, we suggest that whereas soluble penton base proteins may enter cells through clathrinand non-clathrin-mediated pathways, vesicle escape and nuclear delivery appear to be supported by a clathrin-mediated pathway. As our previous efforts have focused on utilizing recombinant penton base proteins as delivery agents for therapeutics, these findings allow us to evaluate the use of the penton base as a cell entry and intracellular trafficking agent, and may be of interest concerning the development of vectors for efficient delivery of therapeutics to cells. Gene Therapy (2006) 13, 821-836.

Novel Fiber-Dependent Entry Mechanism for Adenovirus Serotype 5 in LacrimalAcini

“…Uptake experiments analyzed by confocal fluorescence microscopy showed that after 60 min, much of the membraneassociated penton base protein in HeLa cells had accumulated in the perinuclear region (Fig. 2B); we have previously shown that this pattern of accumulation in HeLa cells is intracellular (41). This incubation time course was also sufficient for internalization of intact Ad5 in LGAC (Fig.…”

“…Recombinant knob, GFP-knob, penton base, and rab3DQL proteins were produced from Escherichia coli as His 6 -tagged fusion proteins and purified from bacterial lysates using Ni-NTA resin as described previously (25,32,33,34,41). Recombinant fiber protein and control protein (glutathione-S-transferase [GST]) were produced from a baculovirus expression system as described previously (40).…”

“…We have also reported that the recombinant penton base protein of Ad5 appears unable to enter LGAC (25), although it can recapitulate the Ad5 entry pathway in HeLa cells (41). Since Ad5 infects LGAC at high efficiency in an apparently pentonindependent manner, we hypothesized that Ad5 might be internalized in LGAC through a unique mechanism.…”

Toward Development of Artificial Viruses for Gene Therapy: A Comparative Evaluation of Viral and Non‐viral Transfection

“…Many viruses, including AAV, Ad, coxsackievirus B, Semliki Forest, hepatitis C, and influenza, require trafficking to and acidification of the late endosomal compartment for effective transduction. 34,35,37,39,40,[77][78][79] It is thought that processing of the viral capsid in an acidic environment may be necessary to activate viral components required for downstream processing, such as nuclear translocation. 77,78 Cell-line transduction dependence also correlates with intracellular trafficking: AAV viruses trafficked through the endo-lysosomal pathway mediate transduction in 293 cells; in contrast, they escape to the cytosol prior to acidification in NIH 3T3 cells, presumably through a different pathway, and are transfection incompetent.…”

“…78 A separate study using an adenoviral protein as a non-viral vector demonstrates internalization through clathrin-dependent and independent processes (the latter likely caveolin-dependent), but vesicle escape and nuclear localization occurs only when the complexes are internalized through processes leading to endo-lysosomal trafficking. 79 Although further research is required to elucidate the mechanisms involved, mounting evidence suggests that both viral and non-viral vectors require processing through the acidified endo-lysosomal pathway for efficient transgene expression.…”