Recombinant EBLV-1 from Clones Cdna
Supplementary Material: Introduction of a genetic marker into the EBLV-1 full length cDNA clone. Prior to the insertion of a NheI restriction site in the 5’-noncoding sequence of the P gene as a genetic marker, non-viral NheI and NotI restriction sites between the CMVie promoter and the downstream hammerhead ribozyme sequences were removed by NheI and NotI digestion of pCMV EBLV-1, blunt end generation and religation. For the insertion an NheI restriction site, the P ORF was RT-PCR amplified with the primer pair PR21/22 from EBLV-1 RNA and after NheI/NotI digestion, the RT-PCR-product was ligated to P ORF upstream and downstream sequences that were amplified with the primer pairs PR23/PR24 and PR25/26 and digested with NheI or Not I respectively. The ligation product was used as a template for PCR-amplification of a 5 kb DNA-Fragment with the primers P23/P26 comprising the P-gene with the NheI and NotI restriction sites in the P gene 5’ and 3’ noncoding sequences. The PCR product was ScaI/BstBI digested and a 4.1 kb DNA fragment was inserted in the ScaI/BstBI digested EBLV-1 cDNA full length clone pCMV EBLV-1 to yield pEBLV-1 NheNot. The NotI restriction site was subsequently removed by RT-PCR amplification of a 1.6 kb kb DNA fragment from EBVL-1 RNA and insertion of a 0.43 kb BstBI/SphI cDNA fragment into BstBI/SphI digested pEBLV-1 NheNot to yield pEBLV-1 Nhe.
Supplementary Material: Cloning of the chimeric cDNA-clones pEBLV-1 L16AGP, pEBLV-1 L16GP and pEBLV-1 L16GP/AGP. For the construction of pEBLV-L16GP a 1.7 kb DNA fragment was PCR amplified from pEBLV1 Nhe template DNA with the primers PR27 and PR28. In a second PCR reaction a 1.9 kb DNA fragment was generated with primers PR23 and PR29 from pCMV L16 template (Finke, Granzow, Hurst, Pollin, & Mettenleiter, 2009). Both PCR products were AvrII digested and subsequently ligated to yield the template for a 3rd PCR reaction using the primers PR28/PR23. The resultant 3.6 kb PCR product was ScaI/NheI digested and a 2.8 kb subfragment was ligated to the 12.3 kb DNA fragment of ScaI/NheI digested pEBLV-1 Nhe. For the construction of pEBLV-1 L16GP/AGP a 2.5 kb DNA fragment was PCR amplified from pEBLV1 Nhe template with primers P30 and P31. In a second PCR reaction a 1.9 kb DNA fragment was generated with primers PR32 and P33 from pCMV L16 template. Both PCR products were SacII digested and subsequently ligated to yield the template for a 3rd PCR reaction using the primers P31 / P32. The resultant 4.7 kb PCR product was ScaI/Acc65I digested and a 2.3 kb subfragment was ligated to the 12.7 kb DNA fragment of ScaI/Acc65I digested pEBLV-1 Nhe. For the construction of pEBLV-L16AGP a 11.4 NotI/ScaI DNA fragment from pEBLV-1 L16GP/AGP was ligated to a 3.7 kb NotI/ScaI DNA fragment of pEBLV-1 Nhe to yield pEBLV-1 L16AGP.
Supplementary Material: Expression plasmids for EBLV-1 proteins. For the construction of pCAGGS EBLV-1 N, the N coding sequence was amplified with the primers PR15/PR16. The 1.4 kb PCR product was digested with MfeI/NheI and the N ORF was ligated to the EcoRI/NheI-digested pCAGGS. For pCAGGS EBLV-1 P, a 0.9 kb DNA fragment was generated by RT-PCR with primers PR17/PR18. After EcoRI/NheI digest, a 0.8 kb partial P sequence was ligated to EcoRI/NheI digested pCAGGS, resulting in pCAGGS-EBLV1-PEcoRI. In a subsequent step, the ligation product was digested with EcoRI and a 0.1 kb DNA fragment from the EcoRI/NheI digested RT-PCR product was inserted to yield pCAGGS EBLV-1 P. For pCAGGS EBLV-1 L, a 0.5 kb RT-PCR product encoding the N-terminal part of the EBLV-1 L gene was generated with the primers PR12 and PR19 and was ligated to the EcoRI/NheI digested pCAGGS. The resulting pCAGGS-EBL1part was MluI/NheI digested and a 5.0 kb DNA fragment was ligated to a MluI/NheI 6.1 kb PCR product that was generated from pHaHd1-EBL1-L (suppl. Fig. 2) with the primers PR19/PR20. The resulting pCAGGS EBLV-1 L encoded the complete L protein.
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