C. Gubser and others Fig. 7. Analysis of cellular inflammatory response by flow cyto- metry. (a, c and d) BAL and (b) lung cells were recovered on the indicated day (a, b and d) or at day 7 (c) p.i. (a, b) Lymphoid population was determined by its characteristic forward and side scatter profiles. (c, d) Percentage of cells expressing activation markers CD69+ or CD25+ in CD3+, CD4+ and CD8+ cell populations. Data shown are the mean percentage from five or six mice ± SEM. P values were determined using Student’s t-test and indicate the mean data that were significantly different between mice infected with v176-WT, mice infected with v176-NHA and mice infected with vH1. To obtain sufficient cells in BALs, cells from two mice were pooled. CD25+, was used in a further experiment and showed that at days 7 and 10 p.i. there were also less CD25+ CD4+ and CD25+ CD8+ lymphocytes in BALs of mice infected with v-176WT compared with control groups, although this difference was only significant for CD8+ lymphocytes at day 7 and CD4+ lymphocytes at day 10 (Fig. 7d). DISCUSSION This paper provides a characterization of the CMLV-CMS 176R gene, which is shown to encode a predominantly 1674 cytoplasmic 57 kDa protein that is expressed both early and late in infection. v-slfn shows sequence similarity to the mammalian schlafen protein family and is most closely related to the short members, m-slfn1 and 2. Construction of an inducible cell line that expresses v-slfn showed that, unlike m-slfn1, v-slfn did not inhibit NIH3T3 cell growth. This is probably due to v-slfn lacking a region with sequence similarity to m-slfn1 aa 1–27 that is essential for m-slfn1-mediated inhibition of fibroblast cell growth (Geserick et al., 2004). The m-slfn proteins are expressed differentially in activated T cells and macrophages, and regulate T-cell development and differentiation (Schwarz et al., 1998; Geserick et al., 2004). Since there is no established small animal model for studying CMLV pathogenesis, we screened several VACV strains, hoping to identify a strain that expresses a full- length v-slfn. However, none of the 16 strains examined expressed a v-slfn orthologue suggesting that, like VACV strain COP, the v-slfn ORF is broken into smaller fragment in these viruses. Sequence data from VACV strains WR, MVA and Lister are consistent with these observations. Therefore, the function of v-slfn in vivo was investigated using a recombinant VACV expressing untagged v-slfn (v176WT) or v-slfn with a C-terminal (v176-CHA) or N- terminal (v176-NHA) HA tag. Expression of WT or tagged v-slfn by recombinant VACV did not affect virus virulence in the intradermal model of infection. However, expression of WT v-slfn or C-terminally tagged v-slfn attenuated VACV in a murine intranasal model, resulting in reduced weight loss and more rapid recovery compared with control groups. In contrast, N-terminally tagged v-slfn did not attenuate VACV in this model, showing that v-slfn function was affected by addition of an HA tag on the N terminus, and suggesting that this part of the protein is important for function. The altered function of N- terminally tagged v-slfn is possibly due to cleavage of a small part of the N terminus of the protein after addition of the HA tag, as shown by immunoblotting (Fig. 3c). v-slfn- mediated attenuation in vivo was characterized by a significantly reduced virus titre in lungs at day 7 p.i. compared with vH1 and v-176NHA, although at day 3 the titres were equivalent. This shows that expression of v-slfn did not prevent establishment of infection or virus replication, but probably accelerated virus clearance by the immune system. Consistently, v176-WT spread to the spleen was delayed and v176-WT was cleared more rapidly from this organ compared with control groups. Characterization of the cellular inflammatory response in the lungs showed that v176-WT infection was characterized by a more pronounced recruitment of lymphocytes that was most striking at day 7 p.i. However, in lung cells, no particular lymphoid subset (CD4+ or CD8+ T cells, B cells or NK cells) was solely responsible for this increase. The mechanism by which v-slfn promotes such vigorous lymphocyte recruitment is under investigation. At day 7 p.i., when mice infected with v176-WT had almost recovered, but mice infected with vH1 and v176-NHA were Journal of General Virology 88