MVA-B DC6L deletion mutant was free of contamination with mycoplasma or bacteria. PCR analysis of MVA-B DC6L deletion mutant To test the purity of MVA-B DC6L deletion mutant, viral DNA was extracted from DF-1 cells mock-infected or infected at 2 PFU/cell with MVA, MVA-B or MVA-B DC6L. Primers RFC6L-AatII-F and LFC6L-BamHI-R (described above) span- ning C6L flanking regions were used for PCR analysis of C6L locus. The amplification protocol was previously described [17]. PCR products were resolved in 1% agarose gel and visualized by ethidium bromide staining. The C6L deletion was also confirmed by DNA sequence analysis. Expression of HIV-1BX08 gp120 and HIV-1IIIB Gag-Pol-Nef proteins by MVA-B DC6L deletion mutant To test the correct expression of HIV-1 proteins HIV-1BX08 gp120 and HIV-1IIIB Gag-Pol-Nef (GPN), monolayers of DF-1 cells were mock-infected or infected at 2 PFU/cell with MVA, MVA-B or MVA-B DC6L. After 24 hours, cells were lysed in Laemmli buffer, cells extracts were fractionated in 12% SDS- PAGE and analyzed by Western blot using rabbit polyclonal anti- gp120 antibody against IIIB (Centro Nacional de Biotecnolog´ıa; 1:3000) or polyclonal anti-gag p24 serum (ARP 432, NIBSC, Centralised Facility for AIDS reagent, UK; 1:3000) followed by anti-rabbit-HRP (Sigma; 1:5000) to evaluate the expression of gp120 and GPN proteins, respectively. Analysis of virus growth To determine virus-growth profiles, monolayers of DF-1 cells grown in 12-well tissue culture plates were infected in duplicate at 0.01 PFU/cell with MVA-B or MVA-B DC6L. Following virus adsorption for 60 min at 37uC, the inoculum was removed. The infected cells were washed once with DMEM without serum and incubated with fresh DMEM containing 2% FCS at 37uC in a 5% CO2 atmosphere. At different times post-infection (0, 24, 48 and 72 hours), cells were collected, freeze-thawed three times and briefly sonicated. The intracellular viruses were titrated by immunostaining as described above. Expression of C6 protein in E. coli and production of anti- C6 polyclonal antibodies The C6 ORF (MVA 019L, 157 aa, 18.2 kDa) was amplified by PCR using primers C6L-NheI-F (59-AGGCTAGCGTTTAGG- AAAAAAAAATATC-39) (NheI site underlined) and C6L-BamHI-R (59-AAGGATCCCATGAATGCGTATAATA-39) (BamHI site under- lined), and VACV MVA DNA as template. The product (488 bp) was digested with NheI and BamHI and cloned into plasmid pET- 27b(+) (Novagen). The ligation product was used to transform BL21 E.coli, and the plasmid of a kanamycin-resistant positive colony was sequenced to confirm that it contained the C6L sequence. The plasmid generated was termed pET-27b-C6L (5837 bp). Plasmid pET-27b(+) provided a tract of 6 histidines at the carboxyl terminus of the C6 protein, generating a recombinant C6 protein of about 24 kDa. Kanamycin-resistant colonies were grown in Luria broth medium until an OD of 0.5 at 595 nm. Isopropyl 1-thio-b-D- galactopyranoside was added (0.5 mM) and the culture grown for 4 additional hours. Cells were pelleted by centrifugation. For lysis, cells were suspended in 50 mM Tris–HCl, pH 7.5, 0.3 M NaCl, 8 M Urea, and incubated with 1 mg/ml lysozyme for 30 min in the presence of 1 mM phenylmethylsulfonyl fluoride. The suspension was freezed-thawed twice. Cellular debris was removed by centrifugation, and the supernatant was incubated with Probound C6L Deletion in MVA-B Improves HIV Immunogenicity resin (Invitrogen). Elution was carried out with different concen- trations of imidazol (100 to 500 mM) in 50 mM Tris–HCl, pH 7.5, 0.3 M NaCl. Eluted fractions were pooled and loaded on desalting columns following the manufacturer’s protocol (GE-Healthcare, Freiburg, Germany), and fractions were collected. Protein was quantified using the Bradford assay, fractionated by 12% SDS- PAGE and analyzed by Western blot using anti-His tag antibody (1:5000) to detect the presence of VACV C6 protein. Fractions containing the C6 protein (with an estimated purity of 90%) were stored in aliquots at 220uC. The C6 protein (1150 mg) was injected into New Zealand White rabbits to produce anti-C6 serum and rabbit polyclonal antibody against C6 (Biomedal Laboratories, Sevilla, Spain). Early expression of C6 protein in cells infected by WR and MVA DF-1 cells were infected with WR and MVA at 5 PFU/cell in the presence or absence of 40 mg/ml of cytosine arabinoside (AraC; an inhibitor of viral DNA replication and therefore late protein expression). At different times post-infection (30 min, 3 h, 6 h and 22 h) cells were lysed in Laemmli buffer, cells extracts fractionated by 12% SDS-PAGE and analyzed by Western blot using rabbit polyclonal sera anti-C6 (Biomedal; 1:100), followed by anti-rabbit-HRP (Sigma; 1:5000). Expression of C6 protein was analyzed also 22 h post-infection in DF-1 cells infected at 5 PFU/ cell with MVA-B and MVA-B DC6L. Immunofluorescence DF-1 cells were grown on glass coverslips (borosilicate glass; BDH) in 12-well plates and infected at 0.5 PFU/cell with WR, MVA, MVA-B or MVA-B DC6L. At 18 h post-infection cells were washed three times in ice-cold PBS, fixed in 4% para- formaldehyde, permeabilized with 0.1% Triton X-100 (Sigma), and blocked with 10% FCS. Cells were incubated with antibodies to the VACV C6 viral protein (rabbit polyclonal anti-C6, 1:500), and A27L viral protein (mouse monoclonal antibody C3anti-14K, 1:400), together with the DNA-staining reagent DAPI (1:200) at RT for 1 h. Samples were incubated with the secondary antibodies (Alexa-488 Goat anti-rabbit IgG and Alexa-546 Goat anti-mouse IgG; 1:500) at RT for 1 h, washed and mounted in ProLongTM Antifade Kit medium. Images were obtained with a Bio-Rad Radiance 2100 confocal laser microscope. RNA analysis by quantitative real-time polymerase chain reaction Total RNA was isolated from THP-1 cells and moDCs infected with MVA, MVA-B and MVA-B DC6L, using the RNeasy kit (Qiagen, Hombrechtikon, Switzerland). Reverse transcription of 100 ng to 500 ng of RNA was performed using the ImProm II RT System kit (Promega). Quantitative PCR was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems, Rotkreuz, Switzerland) using the Power SYBR Green PCR Master Mix (Applied Biosystems), as previously described [40]. Expression levels of IFN-b, IFIT1, IFIT2, RANTES, MIP-1a, IL-8, IP-10 and HPRT genes were analyzed by real-time PCR using specific oligo- nucleotides (sequence be provided upon request). Gene specific expression was expressed relative to the expression of HPRT in arbitrary units (A.U.). All samples were tested in duplicates. IFN-b and type I IFN measurement IFN-b concentrations in cell-culture supernatants were mea- sured by ELISA (PBL Biomedical Laboratories, Picataway, NJ). Type I IFNs were quantified as previously described using the PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e24244