Pdsred1 n1

The construction of expression constructs for pUL97 in wild-type and mutant versions, such as pcDNA-UL97-F, pcDNA-UL97(K355M)-F [30 (link)], and pcDNA-UL97(1-595)-F [18 (link)], has been described elsewhere. To generate C-terminally truncated versions of pUL97 (1-706, 1-702, 1-692, 1-657), fragments were amplified from the template pcDNA-UL97-F [30 (link)]. For amplification primers containing XhoI and EcoRI restriction sites (synthetic primers purchased from Biomers GmbH, Ulm, Germany) were used and PCR was performed with Vent DNA polymerase (New England Biolabs, Ipswich, MA, USA) under standard conditions [35 (link)]. PCR products were subsequently inserted into pcDNA3.1+ (Invitrogen, Carlsbad, CA, USA). A commercially available construct pDsRed1-N1 (BD Clontech, Mountain View, CA, USA) expressing red fluorescent protein was used as a control.

Expression plasmids coding for several tags (HA, AU1, Flag and His) of HCMV pUL50 or pUL53 were generated by PCR amplification of the UL50 or UL53 open reading frame (ORF), using the template pCM1029 [29 (link)]. In addition to individual full-length UL50 or UL53, a fusion version (UL50::UL53), encoding aa 1-397 of UL50 and aa 1-376 of UL53, was also generated. Primers containing tag sequences were also amplified via PCR, resulted in the fusion of the ORFs to C-terminal with different tags. Vent DNA polymerase (New England Biolabs, Ipswich, MA, USA) was obtained for performing PCR with 36 cycles (denaturation for 40 s at 94 °C, annealing for 40 s at 58 °C and polymerization for 90 s at 72 °C). PCR products were cleaved with the restriction enzymes EcoRI/XhoI and were inserted into the vector pcDNA3.1 (Invitrogen). The plasmids coding of HCMV pUL50 or pUL53 expressing red (RFP) or green fluorescent protein (GFP) were inserted into vectors pDsRed1-N1 or pEGFP-N1 (both BD Clontech), respectively after cleavage with the restriction enzyme EcoRI/BamHI. Oligonucleotide primers used for PCR were purchased from Biomers (Supplementary MaterialsTable S1).