C. Gubser and others of recombinant viruses was confirmed by PCR (data not shown). HeLa cells were mock-infected or infected with vDB8R, vH1, v176-WT, v176-NHA or v176-CHA, and expression of v-slfn was detected using a-v-slfn Ab. Fig. 3(c) shows that v176-WT, v176-CHA and v176-NHA express v- slfn whereas, vDB8R and vH1 do not (top panel). Infection was confirmed by detection of the VACV F13 orthologue in all samples in parallel blots (Fig. 3c, bottom panel). The blots were then stripped and reprobed with an a-HA mAb (Fig. 3c, middle panel). This detected the HA-tagged protein expressed by v176-CHA and 176-NHA. There was, however, a reproducible large difference in level of detection between the two samples, suggesting that the N terminus of N- terminally tagged v-slfn is removed by proteolytic cleavage. To determine whether expression of v-slfn altered virus replication, the growth properties of vDB8R, vH1, v176- WT, v176-NHA and v176-CHA were analysed. After infection of BS-C-1 cells at 10 p.f.u. per cell for 24 or 48 h, the total virus yield of all recombinant viruses was indistinguishable (Supplementary Fig. S1, available in JGV Online). Similarly, VACV plaque morphology and size were unaltered by expression of v-slfn in BS-C-1 (Supple- mentary Fig. S2), RK13 and TK2143 cells (data not shown). Therefore, v-slfn does not affect virus growth or plaque size when expressed by recombinant VACV under the condi- tions tested. Subcellular localization of v-slfn To investigate the subcellular localization of v-slfn, HeLa cells were either mock-infected or infected with CMLV strain CMS, VACV WR or v176-CHA for 6 h (Fig. 4). v-slfn was detected as a predominantly cytoplasmic protein with no obvious localization to any organelles (Fig. 4c–e) and similar localization was detected at later time points (data not shown). No signal was present in mock-infected cells (Fig. 4a), and only a weak background was present in cells infected with VACV WR (Fig. 4b). The specificity of the v- slfn antiserum was confirmed by co-staining cells infected with v176-CHA with a-v-slfn Ab (Fig. 4d) and a-HA mAb (Fig. 4e) and merging the images (Fig. 4f), showing co- staining with these two Abs. v-slfn localization was also analysed in NIH3T3 cell lines expressing v-slfn using an a- FLAG mAb (data not shown), and after transient transfec- tion of HeLa cells with the mammalian expression vector pCI alone or pCI expressing v-slfn with or without an HA tag. v-slfn was visualized with either a-v-slfn antiserum or a- HA mAb. All approaches showed the same predominantly cytoplasmic localization for v-slfn, ruling out the possibility that other factors associated with virus infection contributed to v-slfn localization (data not shown). Expression of v-slfn attenuates VACV in the murine intranasal model The virulence of recombinant VACV that does or does not express v-slfn was examined in the murine intradermal and 1672 Fig. 4. Subcellular localization of v-slfn. HeLa cells were either (a) mock-infected or infected with 5 p.f.u. per cell of (b) VACV WR, (c) CMLV-CMS or (d–f) v176-CHA for 6 h and were analysed by immunofluorescence using a-v-slfn (a–d) or a-HA (e) Abs. (f) Shows a merged image of (d) and (e). Bars, 20 mm. intranasal models of infection. In the intradermal model, no difference in lesion size was observed between recom- binant VACV expressing v-slfn or not (Supplementary Fig. S3, available in JGV Online). In the murine intranasal model of infection, mice infected with 107 p.f.u. vH1, v176- WT, v176-NHA or v176-CHA all showed loss of body weight (Fig. 5a, upper panel) and signs of illness (Fig. 5a lower panel); however, from day 4 p.i. there was a significant attenuation (P,0.05) of v176-WT and v176- CHA compared with vH1 and v176-NHA. In addition, mice infected with v176-WT or v176-CHA started to recover sooner after infection (day 6, Fig. 5a) compared with mice infected with vH1 and v176-NHA. This showed that (i) expression of v-slfn attenuated VACV in this model, and (ii) the function of v-slfn was affected by the HA tag at the N terminus. The expression of the influenza virus H1 HA from the TK locus did not alter VACV virulence in this model (data not shown). Journal of General Virology 88