Journal: Virology Journal

Article Title: The effects of N-terminal insertion into VSV-G of an scFv peptide

doi: 10.1186/1743-422X-3-69

Figure Lengend Snippet: scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto Protran nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.

Article Snippet: The samples were fractionated through SDS polyacrylamide (10%) gels (SDS-PAGE) and transferred to Protran nitrocellulose membranes (Schleicher and Schuell).

Techniques: Expressing, Derivative Assay, Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, Sequencing, Immunodetection, Construct, Generated, Amplification, Modification, Produced, Transfection