C. Gubser and others Fig. 1. CMLV v-slfn is related to members of the mammalian slfn family. (a) Diagrammatic representation of CMLV strain CMS v-slfn (GenBank accession no. AAG37679), VACV- WR B2 (YP_233066) and B3 (YP_233067), and murine slfns m-slfn1 (AAH52869), m-slfn3 (NP_035539) and m-slfn8 (NP_ 853523). Protein sizes (number of aa) are shown on the right. (b) CLUSTAL W (Thompson et al., 1994) alignment of the m-slfn1, 3 and 8 conserved region with the C terminus of v-slfn. Identical residues are shown in black and residues conserved in two or three out of four sequences are shown in light or dark grey, respectively. Amino acid co-ordinates are indicated. (c) Unrooted phylogenetic tree showing the relationship of v-slfn from camel- pox virus (CMLV), cowpox virus (CPXV), monkeypox virus (MPXV), ectromelia virus (ECTV), taterapox virus (GBLV) and m-slfns. Protein sequences were aligned using CLUSTAL W and an unrooted tree was generated based on this alignment using PHYLIP on the European Bioinformatic Institute website (http://www.e- bi.ac.uk/clustalw/). Bootstrap values from 1000 replica samplings and the divergence scale (substitutions per site) are indicated. infection with the intracellular pathogens Brucella (Eskra et al., 2003) and Listeria (Geserick et al., 2004), suggesting a role in host defence against pathogens. This study describes the characterization of CMLV strain CMS slfn-like protein 176 (v-slfn). Bioinformatic analyses showed that v-slfn is most closely related to short m-slfns. However, unlike m-slfn1, v-slfn did not inhibit cell growth when expressed stably in mammalian cells. Using specific antiserum, we showed that v-slfn is a predominantly cyto- plasmic 57 kDa protein that is expressed throughout infection. Screening of 18 OPVs showed that immunologi- cally related proteins of similar sizes are expressed by two cowpox virus (CPXV) strains but not by any of the 16 vaccinia virus (VACV) strains tested. To address whether v-slfn affected OPV virulence, CMLV v-slfn was expressed from the thymidine kinase (TK) locus of an attenuated VACV strain. In vivo, this recombinant virus was attenuated further compared with controls, and induced different recruitment of lymphocytes to the site of infection. 1668 METHODS Cells and viruses. BS-C-1, TK2143 and HeLa cells were grown at 37 uC in a 5 % CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % heat-inactivated fetal bovine serum (FBS; Gibco), 100 U penicillin ml21 (Gibco) and 100 mg streptomycin ml21 (Gibco). The source of VACV strain Western Reserve (WR) vDB8R and CMLV strain CMS was described previously (Gubser & Smith, 2002; Symons et al., 2002). Viral growth curves. The one-step growth kinetics of VACV strains were determined as described previously (Pires de Miranda et al., 2003). Plasmids. CMLV strain CMS genomic DNA was used for PCR- mediated amplification of the v-slfn gene. For construction of recom- binant viruses, haemagglutinin (HA)-tagged or wild-type (WT) v-slfn were cloned into pMJ601 (Davison & Moss, 1990) to create plasmids pMJ601-176RWT, pMJ601-176RNHA and pMJ601-176RCHA. Oligo- nucleotide primers were forward primers 176FW (59-CCCCCG- CTCGAGGCCGCCACCATGGCGATGTTTTACGCACACGC-39) and 176FH (59-CCCCCGCTCGAGGCCGCCACCATGTACCCATA- CGATGTTCCAGATTACGCTGCGATGTTTTACGCACACGC-39), Journal of General Virology 88