Genetailor site directed mutagenesis system kit

All mutations within the UL37 gene sequence were performed using the GeneTailor site-directed mutagenesis system kit from Invitrogen according to the kit manufacturer's instructions. Construction of viral mutants with specific amino acid changes was accomplished in Escherichia coli by using the markerless two-step Red recombination mutagenesis system and synthetic oligonucleotides implemented on the bacterial artificial chromosome (BAC) plasmid pYEbac102 carrying the HSV-1(F) genome (a gift from Y. Kawaguchi, University of Tokyo, Japan). Construction of the HSV-1 mutant virus DC480, which has a 12-amino-acid protein C epitope tag inserted immediately after amino acid 480 of HSV-1 UL37, was described previously (26 (link)). The recombinant mutant virus YE102-VC1 (VC1) was modified to express gK and UL20 genes containing V5 and 3×FLAG antigenic epitopes, respectively, and was described previously (39 (link)). Double Red recombination was also used to construct the mutant DC474-480, which has the four conserved tyrosines at positions 474, 476, 477, and 480 of HSV-1 UL37 changed to alanine, as well as mutant viruses DC474 and DC476 with single (Y-to-A) amino acid changes at positions 476 and 477. All mutated DNA regions were sequenced to verify the presence of the desired mutations in BACs and the absence of any other spurious mutations on the viral genome.

We used the GeneTailor Site-Directed Mutagenesis System kit (Invitrogen, California, USA) to obtain plasmids containing either the c.997G > A (p.V333 M) or c.1210A > G (p.M404 V) cDNA sequences, to be used as expression vectors for in-vitro cellular assays. Mutagenesis was performed using the pCMV6-XL4 circular vector carrying the cDNA sequence of LCAT (OriGene, Maryland, USA). Primers for the c.997G > A variant were: forward 5′-GCAGGACTCCCAGCACCTGGTATGGAAGTATAC-3′ and reverse 5′-ACCAGGTGCTGGGAGTCCTGCCAGGAGGTCACG-3′. Primers for the c.1210A > G variant were: forward 5′-CGGGATACAGCATCTCAACGTGGTCTTCAG-3′ and reverse 5′-GTTGAGATGCTGTATCCCGTGCAGGGGCAGC-3′. After transformation in E. coli DH5αTM-T1® (Thermo Fisher Scientific, California, USA), plasmid DNA was purified using AxyPrep Plasmid Miniprep (Axygen Biosciences, California, USA). Plasmid integrity was verified by enzymatic digestion with FastDigest® SmaI restriction enzyme (Fermentas, California, USA) and visualized on a 1% agarose gel. The presence of both c.997G > A and c.1210A > G variants were confirmed by direct sequencing using specific primers for this vector (VP1.5 and XL39, OriGene, Maryland, USA).