The polyclonal rabbit antiserum TATT3 raised towards the capsid VP1 and VP2/VP3 proteins of MVMp was a generous present of P. Tattersall. Goat polyclonal anti glyceraldehyde 3 phosphate dehy drogenase and rabbit anti STAT1 and STAT2 antibodies, too as mouse monoclonal anti PKR antibody had been from Santa Cruz Biotechnology. The polyclonal rabbit antibody directed against the phospho and isoforms of STAT1 was obtained from Cell Signaling. The polyclonal rabbit antibody specic for phospho STAT2 was from Millipore. The mouse monoclonal antibodies directed towards TLR3 and actin have been from eBioscience and MP Biomedicals, respectively. The synthetic dsRNA poly was from GE Healthcare Europe. For transfection, Lipofectamine 2000 from Invitrogen was utilized. Recombinant mouse beta interferon plus the en zyme linked immunosorbent assay kit for detection of mouse IFN have been both obtained from R&D Systems.
Neutralizing antibodies against mouse IFN and IFN have been produced as previously described and puried through ammonium sulfate precipitation at 45% saturation using standard conditions. Neutralizing titers for 7FD3 and 4EA1 have been 1.240,000 and 1.8,000 against 12 IU of recombinant mouse IFN and IFN 4, respectively. Cell culture and virus production. Mouse A9 and L929 broblasts too as human broblastic inhibitor PS-341 NB324K cells have been maintained in minimum essential medium supplemented with 5% heat inactivated fetal bovine serum, 2 mM L glutamine, 100 g/ml penicillin, and 10 units/ml streptomycin. 293T cells were grown in Dulbeccos modied Eagles medium containing 10% FBS and appropriate antibiotics. Low passage primary MEFs freshly isolated from 12. 5 to 13. 5 day postconception embryos of C57BL/6 or CD1 mice had been cultured in DMEM containing 10% heat inactivated FBS with antibiotics.
The multiplicity of infection is expressed as PFU per cell. Primary stocks of wild type MVMp have been produced at the Virus Production Unit of the DKFZ, by calcium phosphate transfection of 293T cells with the pdBMVp infectious mo lecular clone of MVM supplier endo-IWR 1 as previously described. Cells were harvested three days posttransfection, and viruses had been collected by repeated cycles of freezing and thawing in vTE. Crude cell extracts were then applied to reinfect human NB324K cells for a single further amplication of the stock. After subjecting infected NB324K cells to another series of free thaw cycles in vTE buffer, virus stocks have been puried by nonionic iodixanol gradient centrifugation. Viral stocks titers have been determined by plaque assays on human NB324K cell monolayers infected with serial dilutions of virus and are expressed as PFU/ml. Cell transfection with the synthetic dsRNA poly.