Cell entry and trafficking of human adenovirus bound to blood factor X is determined by the fiber serotype and not hexon: heparan sulfate interaction

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

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

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

Improved Adenovirus Type 5 Vector-Mediated Transduction of Resistant Cells by Piggybacking on Coxsackie B-Adenovirus Receptor-Pseudotyped Baculovirus▿

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BVCAR) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BVCAR to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BVCAR-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BVCARGFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BVCAR-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BVCAR to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BVCAR-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BVCAR in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BVCAR-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BVCAR-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BVCAR-Ad5GFP complex. Various situations in vitro or ex vivo in which our BVCAR-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed

Electron microscopy of CHO-K1 cells (a–d) incubated with HAdV5F35 at 10,000 vp/cell in the presence of FX (8 µg/ml), and harvested after 2 h at 37°C.

<p>(<b>a</b>), Representative CHO-K1 cell section showing a cytoplasmic vesicle containing abundant electron dense material. (<b>b–d</b>), Cell surface-bound HAdV5F35 particles. (<b>e</b>), CHO-CD46 cells incubated with HAdV5F35 in the absence of FX (w/o FX). Note the difference in size and sharpness of the viral contour between HAdV5F35 particles seen in (e) and in (b–d).</p

Cellular uptake and extracellular release of HAdV5wt and HAdV5F35 vectors by CHO-K1 cells.

<p>(<b>A</b>), Cell attachment of vector (MOI 5,000) was performed at 4°C for 1 h, and cellular internalization at 37°C for 1 h, respectively, with or without FX (8 µg/ml), as indicated on the <i>x</i>-axis. The number of viral genome copies was determined by qPCR of the fiber gene, normalized to the ß-actin gene. (<b>B</b>), Extracellular vectors associated with microvesicles (MVs) or exosomes (EXOs) recovered from the extracellular medium at 72 h post transduction, were determined as above.</p

Electron microscopy of CHO-K1 cells incubated with HAdV5wt at 10,000 vp/cell, (A) in the absence (w/o), or (B) presence of FX (8 µg/ml) for 2 h at 37°C.

<p>(<b>A</b>), (i) and (ii): General views of cell sections showing (i) intravesicular and (ii) cytoplasmic vector particles. In (iii) and (iv), a vector particle (Vir) is seen within a clathrin-coated vesicle (CCV); (iv), enlargement of the CCV shown in (iii), with measurements of the space between the vector particle and the inner leaflet of the vesicular membrane. N, nucleus; NPC, nuclear pore complexes viewed in a tangential section. (<b>B</b>), (i): vector particle within an endocytic vesicle in the vicinity of a nuclear pore; (ii), viral core seen in the process of traverse of the nuclear pore.</p

Transduction of CHO-K1 or CHO-2241 cells by GFP-expressing, fiber mutants of HAdV5-based vectors in the absence (w/o) or presence of (with) FX (8 µg/ml).

<p>(<b>A</b>), HAdV5wt, mutants HAdV5F^TTAT and HAdV5Pb^EGD and serotype 35 fiber-pseudotyped HAdV5F35 were all used at MOI 2,500, and transduction efficiency were expressed as arbitrary units (AU), as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018205#pone-0018205-g002" target="_blank">Fig. 2</a>. (<b>B</b>), Dose-response effect of FX on cell transduction by HAdV5F^TTAT mutant. CHO-K1 cells were transduced by GFP-expressing HAdV5F^TTAT mutant vector in the presence of increasing concentration of FX (FX_max = 8 µg/ml). Results were expressed as relative transduction efficiency (RTE; refer to the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018205#pone-0018205-g002" target="_blank">Fig. 2</a>).</p

SPR analysis of the <i>in vitro</i> binding of chimeric HAdV5F35 vector to (A) surface-immobilized FX, or (B, C) immobilized HS with or without FX or FXGL.

<p>(<b>A</b>), Representative sensorgrams for HAdV5wt vector (discontinuous lines) injected at 2×10⁹ and 4×10⁹ vp/ml, or for HAdV5F35 injected at the same doses (solid lines). (<b>B</b>), Comparison of binding to HS of HAdV5wt and HAdV5F35 vector particles (2×10⁹ vp/ml) in the presence of FX or FXGL at 720 copies per vector particle. Controls shown are FX and FXGL alone. For better clarity, the sensorgrams for virions alone, which superimposed those of FX and FXGL, are not shown (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018205#pone-0018205-g001" target="_blank">Fig. 1C</a>). (<b>C</b>), Dose-response effect of FX on HAdV5F35 binding to immobilized HS. Note that a detectable signal was observed for 120 copies of FX per virion, and reached the maximal value for 480 copies/vp.</p

SPR analysis of the pH-dependence of FX∶hexon protein interaction.

<p>FX was immobilized on the sensorchip, and a hexon protein solution in 150 mM NaCl, 0.05 M sodium phosphate buffer of various pH values was injected into the flowcell.</p

Cell transduction of CAR- and CD46-negative CHO cells by HAdV5wt in the absence of presence of FX.

<p>(<b>A</b>), Dose-response effect of FX on cell transduction. CHO-K1 cells were transduced by GFP-expressing HAdV5wt vector in the presence of increasing concentration of FX. Both FX_max and FXGL_max corresponded to 8 mg/ml. Results were expressed as relative transduction efficiency (RTE). Transduction efficiency, in arbitrary units (AU), was given using the formula∶TE = (percentage of GFP-positive cells)×(MFI). The RTE was calculated using the formula∶RTE = (TE with FX)∶(TE without FX), with the 1-value attributed to TE in the absence of FX. (<b>B</b>), CHO-K1 (double CAR- and CD46-negative cells) and CHO-2241 (triple CAR- , CD46-, and HSPG-negative cells) were transduced by HAdV5wt vector at MOI 2,500 (left half of the bar graph) or MOI 5,000 (right half of the bar graph) in the absence or presence of FX (8 µg/ml). Results were expressed as RTE, with the 1-value attributed to the TE of CHO-K1 in the absence of FX.</p