Fig. 3. Characterization of v-slfn by immunoblotting. (a) HeLa cells were either mock-infected (M) or infected with CMLV strain CMS at 5 p.f.u per cell and harvested when indicated. + indicates that AraC was present throughout infection. Cell extracts were analysed by immunoblotting using either a-v-slfn or a-F13 Abs. (b) Expression of v-slfn protein in OPVs. Monolayers of BS-C-1 cells were mock-infected or infected with 5 p.f.u. of the indicated virus per cell, harvested at 6 h p.i. and cell extracts were analysed by immunoblotting using either a-v-slfn or a-F13 Abs. CMLV, camelpox virus; VACV, vaccinia virus; WR, Western Reserve; Patw, Patwadangar; King’s Ins, King’s Institute; Tashk, Tashkent; COP, Copenhagen; CPXV, cowpox virus; EP2, elephantpox virus- 2; BPXV, buffalopox virus; RPXV, rabbitpox virus. (c) HeLa cells were either mock-infected or infected with 10 p.f.u. of the indicated VACV per cell, harvested 16 h p.i. and cell extracts were analysed by immunoblotting. In all panels, the positions of molecular size markers (kDa) are indicated. http://vir.sgmjournals.org Camelpox virus schlafen protein The higher level of expression of v-slfn in the absence of AraC indicates that the protein continues to be made after DNA replication has started. In contrast, a 37 kDa protein, which is the orthologue of VACV late protein F13, was not expressed in the presence of AraC (Fig. 3a). Sequence data predict that a v-slfn is expressed by some OPVs; however, the v-slfn ORF is broken in sequenced VACV and VARV strains (http://www.poxvirus.org). To analyse whether other VACV and CPXV strains express a full-length v-slfn, BS-C-1 cells were infected with 16 strains of VACV and two strains of CPXV, and cell extracts were analysed by immunoblotting (Fig. 3b). This showed that none of the VACV strains examined expressed a protein detected by the a-v-slfn Ab. However, in accord with available sequence data, v-slfn was detected in cells infected with CPXV strain Brighton Red (BR). Similarly, elephant- pox virus (EP2), another CPXV strain, also expressed a v-slfn counterpart. For both CPXV strains, the proteins migrated slightly faster than CMLV v-slfn, despite having a comparable predicted molecular mass (CPXV strain BR; http://www.poxvirus.org). Both CPXV v-slfns were detected at lower levels (Fig. 3b); however, this might be due to a-v-slfn Ab specificity. The recognition of a 37 kDa protein (VACV F13 orthologue) by an F13-specific mAb confirmed that all cells had been infected (Fig. 3b, lower panels). Note that the F13 orthologue in buffalopox virus (BPXV)-infected cells was only detected at later times p.i. (data not shown) and that a non-specific doublet is present in mock-infected and infected cells. Thus, v-slfn is an early and late protein that is encoded by a full-length ORF in several OPVs, but is not expressed by any of the 16 VACV strains tested, suggesting that it is either absent or broken into smaller ORFs, as is the case for VACV strains Copenhagen (COP) and WR. Construction of recombinant VACV expressing CMLV v-slfn A VACV strain expressing full-length v-slfn was not identified, therefore the function of v-slfn was studied using a recombinant VACV expressing the CMLV v-slfn. An attenuated VACV strain lacking the B8R gene encoding the gamma interferon-binding protein (vDB8R) (Symons et al., 2002) was selected as the parent virus to meet biosafety concerns that insertion of the CMLV 176R gene into VACV might increase virulence. In addition, the CMLV 176R gene was inserted into the TK locus of vDB8R to provide a second attenuating mutation (Buller et al., 1985). v-slfn was expressed either without (v176-WT) or with an N- or C-terminal HA tag (v176-NHA and v176- CHA, respectively) under the control of a synthetic early and late promoter. v-slfn expression by these viruses was observed from 2 h p.i., similar to the expression pattern seen in CMLV-infected cells (Fig. 3a and data not shown). A control virus expressing the HA from influenza A/PR/8/ 34 (Bennink et al., 1986) from the same locus and using the same promoter was also constructed (vH1). The genotype 1671