Fig. 1

Construction of high-content VirD-GPCR array. a Subcloning of 335 human GPCR ORFs into the UL27 locus of the HSV-1 genome. After the STOP codons were removed from the 335 available GPCR ORFs, they were subcloned into the UL27 locus in the HSV-1 genome on a BAC vector, resulting in fusion with a V5 tag at their C-termini (middle panel). After bacterial transformation, colony PCR reactions were carried out and the products examined using electrophoresis to identify the correct construct (right panel). b Production of VirD-GPCR virions and VirD array fabrication. Confirmed recombinant virus constructs were individually transfected to Vero cells and the viruses were harvested ~7 days post-transfection. Anti-V5 mAb was used to examine expression of the GPCRs as a quality control. Passers were next used to infect cells for virion production. After sucrose cushion centrifugation, a fraction of purified virions was examined again with anti-V5. 315 VriD-GPCRs passed this quality control step and were spotted onto a glass slide to form VirD-GPCR array. The quality of VirD-GPCR array was examined using anti-gD mAb, followed by a Cy3-labeled anti-mouse IgG antibody. All the ViP.rD-GPCRs on the array showed positive anti-gD signals while the BSA showed the lowest signals