Plxsn

The full-length cDNA from human TRIM5alpha was obtained from RT-PCR amplification of HeLa cells RNA. The TRIM5alpha P479L was obtained by recombinant PCR by exchanging the human TRIM5alpha exon 8 with the corresponding mutated allele from patient #H1. The full-length owl-TRIMcypA cDNA was PCR amplified from plasmid pMIG-TRIMCyp obtained from the NIH AIDS reagents program65 (link). Mafa-TRIMCypA and mamu-TRIMCypA were kind gifts of Dr. Greg Towers68 (link)86 (link). The TRIM-CypNup358 was obtained by recombinant PCR as previously described57 (link). All PCR products were cloned into a retroviral vector containing two C-terminus hemagglutinin (HA) tags derived from pLXSN (Clontech). HEK-293T cells were then independently co-transfected with the pLXSN-based retroviral vectors encoding the various HA-tagged TRIM5 variants along with plasmids expressing MLV Gag-Pol and the vesicular stomatitis virus (VSV) G envelope glycoprotein (pCSIG)87 (link). Forty-eight hours after transfection, the retroviral supernatants were harvested and used to transduce dunni cells grown in the presence of G418 at 2 mg/ml (Invivogen) to select stable expression of TRIM5.

In order to construct pcDNA-neo-FL-RAGE or p-LXSN-neo-FL-RAGE vectors, the human FL-RAGE cDNA (GenBank2 accession no. NM001136) was excised from a pre-existing vector by using EcoRI and XhoI restriction endonucleases (New England Biolabs, MA, USA) and inserted into pcDNA-3 or pLXSN (Clontech, CA, USA) digested with same enzymes. To generate pCAGS-IRES-GFP-FL-RAGE, human FL-RAGE cDNA was excised with XhoI, filled-in to create blunt ends, and subsequentially digested with EcoRI. The purified cDNA was then inserted in pCAGS-IRES-GFP (kindly provided by Vania Broccoli) cut with EcoRI and SmaI (Promega, USA).

Cellular and viral genes were expressed using the retroviral vector pLXSN (Clontech, Palo Alto, CA) or the expression vector pcDNA-3 (Invitrogen). The pLXSN-LMP-1 and the mutants LMP-1AxAxA, LMP-1 378 stop, and LMP-1AxAxA/378 stop constructs have been previously described [41] . The pGL3 basic luciferase reporter (Promega) and pGL3 containing the DOK1 promoter constructs have been described previously [29] (link), The NF-κB super-repressor Δ-IκBα, which lacks the coding sequence of the first 36 N-terminal amino-acids, was kindly provided by Dr Elliot Kieff (Harvard Medical School, Boston, Massachusetts, USA). The expression plasmids pDEST-myc-EBNA1, pSG5-EBNA2, pDEST-myc-EBN3A1, pDEST-myc-EBNA3B, pDEST-myc-EBNA3C were kindly provided by Dr Evelyne Manet (ENS, Lyon, France).