| Products/Services Used | Details | Operation |
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| Gene Synthesis | The gK and SPP constructs used in this study are shown in Figures S2 and S3, respectively. In Figure S2, a schematic diagram of full-length gK with an in-frame c-myc tag at the carboxy terminus is shown. Figure S3 shows a schematic diagram of full-length SPP with an in-frame HA tag and ER retention signal also located at the carboxy terminus as we described previously [16]. gK with c-myc tag was synthesized (GenScript, Piscataway, NJ) and inserted into BamHI site of pcDNA3.1 and sequences were verified with standard dideoxy sequencing at the UCLA Genotyping and Sequencing core. Amaxa nucleofactor kit R (Lonza, Allendale, NJ) was used to transfect 106 HeLa or Vero cells with plasmid DNA cocktail containing both HA-SPP and c-myc-gK in a ratio of 1∶1 in accordance with manufacturer instruction. Protein expression was monitored over 5 days using Coomassie blue protein staining and Western blotting. Antibodies against HA and c-myc (GenScript), were diluted according to manufacturer instruction in the total Western HRP kit (GenScript). Optimum c-myc-gK and HA-SPP expression and recovery was determined to be 48–72 hr post-transfection. | Get A Quote |
Glycoprotein K (gK) is a virion envelope protein of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays important roles in virion entry, morphogenesis and egress. Two-hybrid and pull-down assays were utilized to demonstrate that gK and no other HSV-1 genes specifically binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. SPP dominant negative mutants, shRNA against SPP significantly reduced HSV-1 replication in vitro. SPP also affected lysosomes and ER responses to HSV-1 infection. Thus, in this study we have shown for the first time that gK, despite its role in fusion and egress, is also involved in binding the cytoplasmic protein SPP. These results also sug... More
