and reverse primers 176RW (59-CGCCGCCCCGGGTTAAAATT- TTATAGATGACACCC-39) and 176RH (59-CGCCGCCCCGGGTT- AAGCGTAATCTGGAACATCGTATGGGTAAAATTTTATAGATG- ACACCC-39); restriction sites for XhoI and SmaI, respectively, are underlined. The influenza virus A/34/PR/8 HA gene was subcloned from plasmid pGS63 (Smith et al., 1987) into pMJ601 to create plasmid pMJ601-H1. For bacterial expression, the v-slfn gene was cloned into the pET28a vector (Novagen) without the stop codon to obtain a C-terminal histidine (His) tag, creating pET28-176CHis. Oligonucleotide primers were 176FB (59-CCCCCCCATGGCGATG- TTTTACGCACACGC-39) and 176RB (59-CCCCCCTCGAGAAAT- TTTATAGATGACACCC-39); restriction sites for NcoI and XhoI, respectively, are underlined. For production of stable cell lines, the v- slfn gene encoding a C-terminal FLAG tag was cloned into the T-REx tetracycline-inducible expression vector pcDNA4/TO (Invitrogen) creating pcDNA4/TO-v-slfn-FLAG. Oligonucleotide primers were CMLV176 forward (59-CGCTCTAGAATGGCGATGTTTTACGCA- 39) and CMLV176 reverse (5-CCCAAGCTTTTACTTGTCGTCGTC- GTCCTTGTAGTCAAATTTTATAGATGACAC-39). Underlined and bold nucleotides represent the restriction site for HindIII and FLAG tag, respectively. The fidelity of all PCR-generated DNA sequences was confirmed by sequencing. Generation of v-slfn tetracycline-inducible stable cells. NIH3T3 murine fibroblasts were transfected with linearized pcDNA4/TO-v- slfn-FLAG and pcDNA6/TR vectors (Invitrogen) and cell lines were selected under antibiotic selection with 3 mg blasticidin ml21 and 750 mg zeocin ml21 for 3 weeks. Clone B7 was selected as the line exhibiting no basal v-slfn expression and the highest tetracycline inducibility. Production of rabbit polyclonal antiserum to v-slfn. Plasmid pET28-176CHis was transformed into Rosetta-gami Escherichia coli cells (Novagen) and cultured in Luria–Bertani (LB) medium at 37 uC until the OD600 reached 0.6. Protein expression was induced by the addition of 1 mM IPTG for 3 h at 37 uC. Cells were lysed in BugBuster protein extraction reagent (Novagen) containing protease inhibitor cocktail set III (Calbiochem). The bacterial lysates were sonicated and the insoluble material was removed by centrifuga- tion at 10 000 g for 10 min. v-slfn was purified from insoluble inclusion bodies by washing four times in wash buffer (20 mM Tris pH 7.5, 10 mM EDTA, 1 % Triton X-100). SDS-PAGE and Coomassie blue staining confirmed that v-slfn was the major protein present (data not shown). A polyclonal rabbit antiserum was raised against v-slfn by Harlan Seralabs. To reduce cross- reactivity of the antiserum with denatured mammalian proteins, the antiserum was incubated with HeLa cell proteins. HeLa cells (36108) were lysed using acetone and proteins were collected by centrifugation and dried overnight at room temperature. Antiserum diluted 1 : 10 in PBS was added to the precipitate and incubated with shaking for 48 h. The protein precipitate was removed by centrifugation and filtration, and the supernatant was used for immunoblotting. Immunoblotting. BS-C-1 cells were mock-infected or infected at 5 p.f.u. per cell for the indicated time and lysed in radioimmuno- precipitation assay (RIPA) buffer. Protein samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane and probed with a-v-slfn polyclonal rabbit antiserum (1 : 100), rat monoclonal antibody (mAb) 15B6 against the VACV F13 protein (1 : 100; Schmelz et al., 1994), mouse a-FLAG M2 mAb (1 : 1000; Sigma-Aldrich) or mouse a-HA mAb (1 : 1000; Covance). Secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit, anti-rat and anti-mouse IgG (1 : 2000; Sigma-Aldrich). Bound Ab was detected using Enhanced Chemiluminescence Plus Western blotting detection reagents (Amersham Biosciences). http://vir.sgmjournals.org Camelpox virus schlafen protein Immunofluorescence. HeLa cells were grown on sterile glass coverslips (borosilicate glass; BDH) in six-well plates and intracellular proteins were stained as described previously (Law et al., 2004). Samples were examined with a Zeiss LSM 510 laser scanning confocal microscope. Images were captured and processed using Zeiss LSM Image Browser version 3.2. Primary Abs used were a-v-slfn polyclonal antiserum (1 : 200) and a-HA mAb (1 : 500). Construction of recombinant viruses. VACV vDB8R was used as the parental virus for construction of recombinant VACV expressing WT v-slfn, HA-tagged v-slfn or influenza virus HA from the TK locus. CV-1 cells were infected with vDB8R at 0.1 p.f.u. per cell for 1 h and then transfected with pMJ601, pMJ601-176RWT, pMJ601-176RNHA, pMJ601-176RCHA or pMJ601-H1. Virus was harvested from infected cells 3 days later and recombinant TK2 viruses were selected by growth on TK2143 cells in the presence of 25 mg 5-bromodeoxy- uridine (BUdR) ml21 and chromogenic substrate X-Gal, as described previously (Chakrabarti et al., 1985). Blue plaques were picked, plaque purified and the presence of v-slfn was confirmed by PCR and immunoblotting. Resulting viruses were named vDB8R, v176-WT, v176-NHA, v176-CHA and vH1, respectively. Mouse intradermal and intranasal model of infection. The intradermal inoculations were carried out as described previously (Tscharke & Smith, 1999). For the intranasal model, groups of female BALB/c mice (6–8 weeks old) were infected intranasally with 46106 or 107 p.f.u. sucrose-purified virus in 20 ml PBS, and their weight and signs of disease were scored daily as described previously (Alcami & Smith, 1992). Mice infected with 46106 p.f.u. vH1, v176-WT or v176-NHA were sacrificed at 3, 5 and 7 days post-infection (p.i.). Cells present in the alveoli were removed by bronchoalveolar lavage (BAL) using BAL solution (12 mM lidocaine and 5 mM EDTA in Earl’s balanced salt solution), centrifuged at 800 g, resuspended in erythrocyte lysis buffer (0.829 % NH4Cl, 0.1 % KHCO3, 0.0372 % Na2EDTA) for 3 min and kept on ice in RPMI/10 % FBS. Lung cells were obtained from lung homogenates by enzymic digestion, lysis of erythrocytes and centrifugation through 20 % Percoll (Sigma- Aldrich), as described previously (Clark et al., 2006). Live cells in BAL and lung single-cell suspensions were counted, blocked and stained with appropriate combinations of fluorescein isothiocyanate-, phycoerythrin- or tricolour-labelled a-CD25, a-CD69, a-CD3, a-CD8, a-CD4, B220 or a-DX5 and the relevant isotype mAb controls (BD Biosciences) as described previously (Clark et al., 2006). The distri- bution of cell-surface markers was determined on a FACScan flow cytometer with CellQUEST software (BD Biosciences). A lymphocyte gate was used to analyse data from at least 20 000 events. The titres of virus in tissues were determined as described previously by plaque assay on duplicate monolayers of TK2143 cells (Reading & Smith, 2003). Statistical analysis. Student’s t-test (two-tailed) was used to test the significance of the results. RESULTS CMLV protein 176 (v-slfn) is related to members of the slfn family The characterization of murine slfn1 (m-slfn1), the prototype of the slfn protein family, led to the identifica- tion of related sequences in several OPVs (Schwarz et al., 1998). Subsequent sequencing of CMLV identified another v-slfn orthologue (protein 176, called v-slfn hereafter) (Gubser & Smith, 2002). CMLV v-slfn was predicted to 1669