Journal: Journal of Virology
Article Title: Roles of the Phosphorylation of Herpes Simplex Virus 1 UL51 at a Specific Site in Viral Replication and Pathogenicity
Figure Lengend Snippet: Strategy to construct a self-excisable HSV-1(F) BAC clone. (A) Lines 1 and 2, schematic diagrams of E. coli plasmid pYEbac102 and pYEbac102Cre, respectively, contained in GS1783; line 3, schematic diagrams of recombinant virus YK312 (ΔBAC). (B) Schematic diagram of the genome structures of wild-type HSV-1(F) and relevant domains of the recombinant viruses. Line 1, sequence arrangement of the HSV-1(F) genome. Line 2, location of the EcoRV-HindIII fragment used as a labeled probe in panel C. Line 3, an enlarged portion of the EcoRI J and D fragments of HSV-1(F). Lines 4 and 6, double-headed arrow represents the fragment region. Lines 5 and 7, summary of DNA fragments from recombinant viruses with or without the BAC sequence and the poly(A) signals. (C) Southern blot analysis of EcoRI-digested DNA isolated from YK312 (ΔBAC)- or YK304 (BAC)-infected cells using the EcoRV-HindIII fragment of pRB442 as a probe. The letters on the right refer to the designations of the DNA fragments generated by restriction endonuclease EcoRI cleavage.
Article Snippet: This was then cloned into the EcoRI site of pcDNA3.1(+) (Thermo Fisher Scientific). pcDNA-FEM-KanS was constructed by PCR amplification of the domain containing the FEM tag, I-SceI site, and kanamycin resistance gene from pBS-FEM-KanS and cloning it into the EcoRI and HindIII sites of pcDNA3.1/myc-His(−)-A (Thermo Fisher Scientific). pcDNA-FEM was constructed by digesting pcDNA-FEM-KanS with NheI and self-ligating to remove the I-SceI site and kanamycin resistance gene. pcDNA-FEM-UL51 was constructed by amplifying the entire UL51 open reading frame (ORF) from pYEbac102 by PCR and cloning it into pcDNA-FEM in frame with FEM. pBS1007, used to generate the recombinant virus YK639 (ΔUL51-repair), was described previously ( ). pRB442, used to generate the probe for Southern blotting, contains a 6.3-kbp KpnI-HindIII fragment of HSV-1(F), which encompasses the left end of the UL region, including the region between 0.0415 and 0.084 map units, as described previously ( ).
Techniques: Construct, BAC Assay, Plasmid Preparation, Recombinant, Sequencing, Labeling, Southern Blot, Isolation, Infection, Generated