Distribution of cells infected with VV and/or expressing the SFV replicon in BSC-1 cells.

<p>BSC-1 cell monolayers were infected with dilutions of W-SFR or W-H-SFR for 48 h, fixed and stained with anti-B5 antibody. Fluorescence within and around virus plaques was visualized in an inverted fluorescence microscope. Merged images result from the combination of monochrome images in red (anti-B5 antibody) and green (direct expression of GFP). Images covering whole plaques and tails were assembled from multiple individual images stitched together as described in the Materials and Methods section. White boxes specify the areas inside (plaque) or outside (tail) the virus plaques that are shown enlarged in the smaller photographs.</p

Distribution of cells infected with VV and/or expressing the SFV replicon in BHK-21 cells.

<p>BHK-21 monolayers were infected with dilutions of VV-rsGFP, W-SFR or W-H-SFR. At 48 h.p.i, cell monolayers were fixed and stained with polyclonal antiserum to VV proteins. Merged images result from the combination of monochrome images in red (anti-VV polyclonal serum) and green (direct expression of GFP). The larger panel was assembled using overlapping photographs stitched together as described in the Materials and Methods section.</p

Particle production in the presence of vaccinia virus inhibitors.

<p>Hela cells were infected at a m.o.i. of 5-H-SFR and after adsorption, medium was replaced with fresh normal medium (brown rhombs) or medium containing 100 µg/ml AraC (red squares), 10 µg/ml IMCBH (black triangles) or 100 µg/ml Rifampicin (blue squares). Samples of the culture medium were collected every 12 hours, clarified, filtered and stored at 4°C until last point was collected. To determine the titer of SFPs, fresh BHK cells were infected with serial dilutions of the filtered supernatant, and GFP-positive cells were counted 24 hours later in the fluorescence microscope. <b>Lower panels:</b> Vaccinia virus Extracellular virus (EV) or cell associated (IV) production. Hela cell monolayers were infected at a multiplicity of 5 pfu per cell with W-H-SFR recombinant virus, and after 1 hour medium was replaced with medium containing AraC (100 µg/ml), rifampicin (100 µg/ml) or IMCBH (10 µg/ml). Virus was harvested at 24 hpi and Vaccinia virus titers in the culture medium (EV) (left panel) and cell lysates (IV) (right panel) were obtained by plaquing in BSC-1 cell monolayers and are represented relative to the control with no inhibitor.</p

Plaque formation and SFP production by W-H-SFR.

<p>Plaque phenotype of the viruses indicated was determined in a standard vaccinia plaque assay on BSC-1 monolayers. Virus plaques were allowed to develop for 48 hours and stained with crystal violet solution. In the cultures treated with anti-SFV antibody, culture medium was replaced at 2 hours post-infection with medium containing polyclonal antiserum to SFV proteins (α-SFV).</p

Construction of VV containing a SFV replicon.

<p>Upper panel: Schematic representation of the virus genome, indicating the F13L and the TK loci. Viruses W-RednsTK and W-H-RednsTK were obtained by recombination of VV WR and VV-Helper, respectively, with plasmid pRednsTK. Viruses W-SFR and W-H-SFR were obtained by recombination of W-RednsTK and W-H-RednsTK, respectively, with plasmid pMix-f70An. Boxes labeled TKL and TKR denote the left and right recombination flanks or the TK gene. Boxes ns1–4* correspond to the genomic region coding the non-structural SFV proteins with an early VV transcription termination signal mutated. Black circle: VV synthetic early/late promoter. dsRed: red fluorescent protein gene. Helper: SFV genes for structural proteins, inserted downstream of the F13L gene under the control of a VV synthetic early/late promoter. rsGFP: green fluorescent protein gene. f: sequence from the 3′end of the SFV replicon, including 70 nucleotides adjacent to the PolyA and a 70 nt PolyA sequence. In the lower right, plaques formed by W-SFR or W-H-SFR on monolayers of BSC-1 cells for 48 hours. Below, sequence of the VV TK promoter and the predicted 5′ end of the SFV replicon (arrow).</p

Coinfection of VV and SFV.

<p><b>A)</b> BSC-1 cells were infected with VV expressing β–Glucuronidase and coinfected at different times with SFPs containing a SFV replicon with the β–Galactosidase gene. Both infections were carried out at a moi of 5 pfu/cell. 48 hours after the first infection, cells were lysed, and the β-galactosidase and β-glucuronidase activities in extracts corresponding to 10³ cells were assayed. <b>B)</b> BSC cells were infected with VV and then infected with SFV at different times post-vaccinia infection. At 48 hours, titers of Vaccinia virus were obtained by plaquing lysed cells (grey bars), and SFV titers were determined from culture media (black bars).</p

Electron microscopy of W-H-SFR infected cells.

<p>Vaccinia virus (particles around 200–250 nm) and smaller, SFV-like particles (SFPs, spherical, approximately 50 nm diameter) were present in W-H-SFR infected cells (Panels A-E). Note that SFPs tend to aggregate in clusters (central panels). A budding image at the plasma membrane can be seen in panel C. An image of the control of W-SFR infected cells (panel F) is shown.</p

Transcription and packaging of the SFV replicon in VV-infected cells.

<p><b>A)</b> BSC-1 cells were infected with vaccinia virus expressing SP6 RNA polymerase (VV-Sp6) and subsequently transfected with plasmid pSFV-LacZ. At 48 hours, cells were either stained for β-galactosidase by addition of X-Gal to the cultures or lysed to assay β-galactosidase activity. Transf.: Number of β-galactosidase positive cell in a 24-well plate well. β-Gal T: β-galactosidase in 10⁵ cells (pg). β-Gal/cel: ratio of β-galactosidase activity per cell. <b>B)</b> Packaging of replicon RNA by SFV structural proteins expressed from V-Helper. BHK-21 cells were transfected with plasmid pSFV-GFP, or with <i>in vitro</i> transcribed RNA from pSFV-GFP linearized with SpeI, and mock infected or infected with V-Helper at a moi of 5 pfu/cell. Column SFPs/ml shows the titers of SFPs in the culture media at 48 h post-infection. C) Western blot analysis of cell extracts infected with the viruses indicated at the top. The positions of SFV mature structural proteins p62, E1 and C are indicated.</p