C. Gubser and others encode a 502 aa protein with an N-terminal domain that has 22 % amino acid identity (approx. 41 % similarity) to an uncharacterized family of baculovirus proteins, collec- tively known as p26 (Liu et al., 1986), and a C-terminal domain related to the conserved region of m-slfns (Fig. 1). v-slfn is poorly conserved amongst chordopoxviruses, with only members of the OPV genus encoding putative v-slfn counterparts; however, a p26-like domain is present in certain entomopoxvirus proteins. Melanoplus sanguinipes entomopoxvirus (MSEV) ORF 237 encodes a protein with 43 % identity and 61 % similarity to the first 197 aa of CMLV v-slfn, although this sequence is absent in Amsacta moorei entomopoxvirus (AMEV) (Afonso et al., 1999; Bawden et al., 2000; Tulman et al., 2006). Other OPVs predicted to encode full-length v-slfns are CPXV, ectromelia virus (ECTV), monkeypox virus (MP- XV) and taterapox virus (GBLV) (http://www.poxvirus. org), whereas in VACV and VARV, the corresponding gene is broken into fragments. All full-length OPV v-slfns share between 84 and 100 % amino acid identity (93–100 % amino acid similarity) with CMLV strain CMS v-slfn. Fig. 1(a) shows a depiction of CMLV v-slfn protein and its relationship with the v-slfn fragments from VACV strain Western Reserve (WR) and a representative member of each of the m-slfn subgroups. Altogether, CMLV v-slfn is most similar to mammalian short m-slfns (m-slfn1 and 2), lacking the C-terminal extensions of intermediate (m-slfn3 and 4) and long (m-slfn5, 8, 9, 10 and 14) m-slfns. Fig. 1(b) shows an alignment of the conserved region of m-slfns with the C terminus of v-slfn. In this region, v-slfn shares between 24 and 30 % identity (42–60 % similarity) with m- slfns. Notably, v-slfn lacks similarity to the first 27 aa of m- slfn1, which are essential for m-slfn1-mediated inhibition of cell growth of fibroblasts (Fig. 1b; Geserick et al., 2004). The unrooted tree in Fig. 1(c) shows the phylogenetic relationships of OPV v-slfn proteins with their mammalian counterparts. Due to their high sequence similarity, all OPV v-slfns group closely together. Compared to mam- malian counterparts, v-slfns are most closely related to short m-slfns (m-slfn1 and 2) and least related to long m- slfns (m-slfn5, 8, 9, 10 and 14), due to their lack of C- terminal extension and also because of a lower degree of similarity within the m-slfn conserved region (Fig. 1b). Fig. 1(c) also highlights the reported divergence of m-slfn5 protein sequence (Geserick et al., 2004) and that of the newly identified m-slfn14 (GenBank accession no. XP_ 899217) to other long m-slfns. Expression of v-slfn in fibroblasts v-slfn is most similar to short m-slfns, of which m-slfn1 inhibits fibroblast cell growth in vitro (Schwarz et al., 1998; Brady et al., 2005). To investigate whether v-slfn had a similar effect on cell growth, a C-terminally FLAG-tagged v-slfn was expressed stably in NIH3T3 cells using the T- REx inducible system (Methods). Two independent clones (clone B7; Fig. 2 and data not shown) were induced for 1670 Fig. 2. v-slfn effect on NIH3T3 cell growth. (a) NIH3T3 cell line (B7) expressing v-slfn stably upon tetracycline induction was seeded at a density of 5 105 cells per 100 mm dish in triplicate and, after 24 h, expression of v-slfn was induced or not by addition of tetracycline (0.5 mg ml”1). Cells were cultured over 72 h, harvested by trypsinization and counted in duplicate. (b) Cell lysates from (a) were analysed for expression of v-slfn using an a- FLAG mAb (arrow). Data are representative of three independent experiments. Positions of molecular size markers (kDa) are indicated. expression of v-slfn and after 24, 48 and 72 h the number of viable cells was assessed (Fig. 2a). Although v-slfn was expressed after addition of tetracycline (Fig. 2b, arrow), there was no difference in cell proliferation between cells that did or did not express v-slfn, showing that unlike m-slfn1, but like intermediate long m-slfns (Schwarz et al., 1998; Geserick et al., 2004; Brady et al., 2005), v-slfn does not affect fibroblast cell growth in vitro. Detection and characterization of v-slfn To characterize v-slfn, the CMLV 176R gene was expressed in E. coli with a C-terminal His tag, and the recombinant protein was purified from inclusion bodies and used to raise a rabbit a-v-slfn polyclonal Ab (Methods). In immunoblots, the a-v-slfn Ab detected an approximately 57 kDa protein in cells infected with CMLV (Fig. 3a, b) or with a recombinant VACV expressing CMLV v-slfn with or without an HA tag (Fig. 3b, c). v-slfn was not present in mock-infected cells. The time of expression of the v-slfn protein during infection was investigated in CMLV-infected cells (Fig. 3a). v-slfn was expressed from 2 h p.i. and its expression levels increased until 16 h p.i. The protein was also detected at 16 h p.i. in cells in the presence of cytosine arabinoside (AraC), an inhibitor of DNA replication and thereby of virus intermediate and late gene expression, indicating that v-slfn was expressed early during infection. Journal of General Virology 88