Cellular Trafficking of FX-Ad5fiber35 Complex but should be taken into consideration in the future design of target tissue-redirected adenoviral vectors. HAdV5 particle titers ranged from 561011 to 161012 vp/ml, with infectious titers between 261010 and 561010 PFU/ml [95]. Materials and Methods Cell lines E1A-E1B-trans-complementing HEK-293 cell line (abbreviated 293; CRL 1573) was obtained from the American Type Culture Collection (Manassas, Va). HEK-293 cells were cultured as monolayers in DMEM supplemented with 10% fetal calf serum (FCS, Sigma), penicillin (200 U/ml), and streptomycin (200 mg/ ml; Gibco-Invitrogen) at 37uC and 5% CO2. The 293-derived, fiber-trans-complementing cell line, abbreviated 293-Fiber, was obtained from Transgene SA (Strasbourg, France). 293-Fiber cells were grown in the same medium as 293 cells, supplemented with hygromycin at 350 mg/ml [70,83]. Chinese hamster ovary cells CHO-K1, and proteoglycan-deficient CHO-2241 cells (pgsB-618, ATCC code CRL-2241) were obtained from the European Collection of Cultured Cells via the Institut de Biologie Structurale, Grenoble, France [79]. CAR-expressing CHO cells (CHO-CAR) were obtained from Dr. J. Bergelson [4], and CD46- expressing CHO cells (CHO-CD46) from Dr. D. Gerlier [84]. CHO-K1, CHO-2241, CHO-CAR and CHO-CD46 cells were cultured as monolayers in Alpha-MEM supplemented with 10% FCS, penicillin and streptomycin as above (Gibco-Invitrogen). All cells were incubated at 37uC under 5% CO2 . HAdV5-based vectors and nomenclature Replication-deficient HAdV5 vectors (E1A–E1B, and E3 deleted) expressing GFP were propagated in trans-complementing cell line 293. Their genetic construction and phenotypes have been described in detail in previous studies [85–93]. Since all the vectors used in the present study contained the same reporter gene coding for the green fluorescent protein (EGFP) cloned downstream to the hCMV promoter, their acronyms did not specify this reporter gene, in contrast to our previous studies. For reason of simplification, their acronyms only referred to the capsid component serotype and to the protein mutated, penton base (Pb) or fiber (F). (i) HAdV5 vector with wild-type (wt) capsid. Control HAdV5 vector with nonmodified capsid was abbreviated HAdV5wt. (ii) Penton base mutant. Mutant HAdV5PbEGD carried a RGD-to-EGD substitution at position 340 in the penton base coding sequence. (iii) Fiber shaft substitution mutant. In the HAdV5FTTAT vector, the KKTK motif at position 91–94 in the fiber shaft domain was modified into 91-TATT-94 using conventional PCR- based mutagenesis [88,91]. (iv) Chimeric fiber vector HAdV5F35. HAdV5F35 carried the HAdV serotype 35 fiber knob and shaft domains fused to the HAdV5 fiber tail [68,85]. Adenovirus purification and titration HAdV5-based vectors were purified by CsCl gradient ultracen- trifugation using conventional methods [85,94]. The infectious titer of the HAdV5 vector stocks was determined by the plaque assay method in HEK-293 or in double trans-complementing cell line 293- Fiber [70] for HAdV5 fiber mutants, and expressed as PFU per ml [91,92]. The titer in physical particles (viral particles; vp) was determined by absorbance measurement at 260 nm (A260) of 1-ml samples of SDS-denatured virions (0.1% SDS for 1 min at 56uC) in a 1-cm-path-length cuvette, using the respective formula: A260 of 1.0 = 1.161012 vp/ml for HAdV5 (genomic DNA = 36 kbp). Fluorescent labeling of adenovirus particles and FX protein Alexa FluorH 488 dye (tetrafluorophenyl ester; Molecular Probes; Invitrogen), and Alexa FluorH 555 dye (succinimidyl ester; Molecular Probes; Invitrogen), were abbreviated Alexa-488 and Alexa-555, respectively. Random labeling of HAdV5wt or HAdV5F35 vector particles with Alexa-488 was carried out as follows. Aliquots of 161012 vector particles in suspension in 0.05 M HEPES buffer pH 7.2 (900 ml, final volume) were incubated with chemically reactive Alexa-488 used at a 20-fold excess over the 18,000 amino groups present at the surface of the adenoviral capsid, for 2 h at room temperature (RT) and in argon atmosphere. The reaction was stopped by adding 100 ml 1 M lysine solution in 0.05 M HEPES buffer pH 7.2, corresponding to a 5-fold excess of lysine over the Alexa-488 reagent. After 1 h incubation at RT, the labeled-vector particles were separated from unreacted dye by gel filtration on a PD10 column. For labeling of FX protein with Alexa-555, FX protein (5 mg/ml) was reacted with 1 mM Alexa FluorH 555 dye in 0.05 M HEPES buffer pH 7.2, for 1 h at RT, and excess of unreacted dye eliminated by dialysis against PBS. Live cell imaging and time-lapse microscopy Samples of CHO-K1 or CHO 2241 cell monolayers (56105 cells/well) were seeded directly onto poly L-lysine-coated glass dishes (Mattek Corp., USA), and maintained in the appropriate medium at 37uC for 24 h. Cells were infected with 35 ml of recombinant baculovirus suspension expressing fluorescent mar- kers of cellular organelles or compartments (CellLightTM Lyso- somes-RFP and CellLightTM Endosomes-RFP *BacMam 2.0*) per cm2 of monolayer in serum-free medium for 4 h at RT, according to the supplier instructions (Invitrogen). Suspension was then withdrawn and replaced by culture medium supplemented with 10% FCS. Cell samples were further incubated overnight at 37uC under 5% CO2 atmosphere, then processed for infection with fluorescent-labeled vector particles, as follows. The following day, the cells were washed with cold PBS (4uC), and overlaid with 1 ml fresh and cold (4uC) medium. Alexa-488-labeled vector particles (10,000 vp/cell, in a volume of 100 ml) were added to the medium, and incubated on ice for 1 h. The cells were then washed 3 times with PBS, followed by fresh and cold (4uC) medium. Cell samples were kept on ice until they were transferred to 37uC in the incubation chamber of the confocal microscope. Observations were performed from 10 min to 3 h pi, using an inverted confocal laser scanning microscope (LSM 510 Meta; Carl Zeiss, IAB), equipped with a 636 oil immersion objective (Plan-Apochromat 636/1.4) and a humidified CO2- and temperature-controlled incubation chamber. The Alexa-488 dye was excited by the Argon laser and the emission was collected with a BP 500–550 nm filter. Orange channel used 543 nm HeNe laser excitation and LP560 emission filter. The images were mainly collected in the focal plane crossing the cell nucleus with a resolution of 5126512 pixels (Zoom 1) and 46 line average. The collection time was 3 s per channel. The pinhole of the fluorescence channels was set to one Airy unit corresponding to the optical section of less than 1 mm and the signal intensity was adjusted with a PMT gain to completely fill the detector dynamic range. The two channels were acquired in a sequential mode to avoid spectral cross-talk, and the acquisition of transmitted light image was concomitant with the green channel. PLoS ONE | www.plosone.org 15 May 2011 | Volume 6 | Issue 5 | e18205