Fig 1.
Construction of virus recombinants by combined positive and negative selection.
A. A vaccinia virus expression plasmid encoding an ORF of interest (ORF-X) is cloned downstream of a bacteriophage T7 promoter (pT7) under the additional transcriptional control of a Lac operator (LacO), an encephalomyocarditis (EMC) virus leader sequence and a bacteriophage transcriptional stop sequence (TT7). A positive selection marker (GPT behind a 7.5 early/late vaccinia virus promoter) is included on the plasmid. Sequences on the left and right sides of the vaccinia virus hemagglutinin gene (HA-L and HA-R) are used in vivo to recombine with the MVA-Koom virus which encodes T7 RNA polymerase, GFP as well as the GYR-PKR and the mCherry-NeoR fusion genes. Replacement of the fusion genes by OFF-X is selected by the ability of the recombinant virus to multiply in the presence of MPA and coumermycin resulting in loss of mCherry expression. Note that the sequences are represented according to the standard orientation of the virus genome. B. Analysis of recombinant virus plaques by light and epifluorescence microscopy. BHK 21 cells were infected in the presence of IPTG with MVA-Koom or an MVA-T7 recombinant virus derived from MVA-Koom by insertion of a polyprotein sequence encoding YFP at its 3’ end (see Fig 4). One representative virus plaque for each infection was observed with an EVOS Cell Imaging System using from top to bottom a white light cube (top panel), a GFP light cube (470/22, 510/42), a YFP light cube 500/24, 524/27 or an RFP light cube (531/40, 593/40).
Fig 2.
Protein production in suspension cell cultures.
A. BHK 21 suspension cells (1.2 106 cells/ml) were infected in 50 ml cell culture medium with MVA-T7 encoding β glucuronidase at increasing MOIs. After 24 hours infection cells were pelleted and enzymatic activity determined in duplicate. B. BHK 21 suspension cells (106 cells/ml) were infected in a 100 ml cell culture medium with an MVA-T7 virus encoding a GST-tagged HIV-1 integrase at 0.1 PFU/cell. After 48 hours infection in the absence of IPTG, 1 ml, 2 ml, 5 ml, 10 ml or 20 ml of infected cells were added to 50 ml of uninfected cells (106 cells/ml). Cell culture medium was added so that the culture volumes were identical in all samples and protein expression was induced by the simultaneous addition of IPTG. Control infections were conducted with 4PFU/cell of the MVA-T7 virus encoding GST-integrase in the presence or absence of IPTG. 24 hours later 1.5ml of the infected cell cultures were recovered for analysis of total protein by PAGE. U uninfected cells.–and + indicate the presence or absence of IPTG in the infected cell cultures. C. 45 ml samples of the infected BHK 21 suspension cultures in panel B were pelleted, sonicated and the GST-integrase purified and examined by PAGE. U uninfected cells.–and + indicates the absence or presence of IPTG in the infected cell cultures.
Fig 3.
Overview of mammalian protein expression in infected cells.
Schematic illustration of the mammalian expression system. The top part of the figure depicts amplification of virus in a preproduction setting in the absence of inducer (IPTG). The bottom part depicts multiplication of uninfected cells for two days followed by addition of infected cells to uninfected cell cultures. Inducer is added upon cell mixing and one day latter infected cells are recovered and purification of protein complexes can be carried out.
Fig 4.
Expression of the IN/LEDGF complex.
A. Schematic drawing of the ORFs inserted into the MVA-T7 virus behind a T7 promoter and encoding a polyprotein including from 5’ to 3’ the TEV protease, LEDGF, two distinct copies of the HIV-1 IN and YFP. Each ORF is separated from the other by twin TEV cleavage sites (perpendicular arrows). B. Purification of the IN-LEDGF complex from 12 109 suspension BHK 21 cells infected with an MVA recombinant encoding the complex. The His-tagged complex was first purified by HPLC on a HisTrap excel Ni sepharose column (not shown) then the pooled fractions were concentrated and separated by size fractionation on a Hi Load 16/60 Superdex-200 column. The position of the B6 peak fraction is boxed in red. C. PAGE was performed on aliquots of the fractions recovered from the Superdex-200 column. Arrows point to the LEDGF and IN proteins stained with Coomassie blue. D. 3’ processing test of the IN/LEDGF complex. The release of GT-fluorescent dinucleotide was monitored by following fluorescence anisotropy as a function of time for the IN-LEDGF complex in the absence of DNA; in the presence of non-specific DNA or in the presence of HIV-1 U5 DNA.
Fig 5.
Expression of the Vif-APOBEC complex.
A. Schematic drawing of the ORFs inserted into the MVA-T7 virus behind a T7 promoter and encoding a polyprotein including from 5’ to 3’ the TEV protease, CBFβ, VIF, EloB, EloC, APOBEC3G and YFP. Each ORF is separated from the other by twin TEV cleavage sites (perpendicular arrows). B. Cytidine deaminase activity in the absence of cell extract (left) or in the presence of cell extracts from BHK21 cells infected with MVA-T7 encoding APOBEC3G (middle) or MVA-T7 encoding the Vif-APOBEC complex (right). Enzymatic activity was determined in a two-step reaction where the appearance of the 17 and 22 oligonucleotide products was indicative of deaminase activity. C. Purification of the Vif-APOBEC complex from 2 liters of suspension BHK 21 C13-2P cells. Cell lysates clarified by ultracentrifugation at 100,000 g were injected onto a 1 ml StrepTactin® column and washed extensively. Peak fractions were combined, concentrated and examined by PAGE (right lane). Molecular weight markers are displayed in the left lane. Arrows point to proteins migrating with the mobility expected for APOBEC3G, CBFβ, Vif, EloB and Elo C. The identity of these proteins was confirmed by mass spectrometry. Proteins were stained with Coomassie blue.