FIG 2.

Generation of a ORF63-deficient MuHV-4 mutant. (A) Schematic representation of the strategy followed to produce the recombinant MuHV-4 strains. The ORF63-deficient MuHV-4 mutant was derived from a cloned MuHV-4 BAC by a galK counterselection method. The ORF63 coding sequence was disrupted by inserting stop codons (ORF63 STOP). The mutation incorporated new BamHI restriction sites. This virus was reverted by homologous recombination with a WT genomic segment (ORF63 Rev). TRs, terminal repeats. (B) Verification of the molecular structure. BAC DNA was digested with BamHI, resolved by agarose gel electrophoresis, and hybridized with a ³²P-labeled probe, corresponding to nucleotides 83819 to 84693 of the MuHV-4 WUMS strain genome. The black arrowhead shows the WT ORF63 fragment (13,724 bp). Open arrowheads show the restriction fragments that contain ORF63 STOP (7,121 bp and 6,627 bp, respectively, for the left and the right fragments). Sizes in kilobase pairs are indicated on the left. (C) Viral transcription in ORF63⁺ and ORF63⁻ viruses. BHK-21 cells were infected with WT, ORF63 STOP, and ORF63 Rev MuHV-4 strains (0.5 PFU/cell), and 24 h later, RNA was extracted, reverse transcribed, and assayed for viral transcripts by qRT-PCR amplification of part of each gene. ORF62 and ORF64 flank the ORF63 gene of MuHV-4. ORF25 is a control viral gene. The data are averages from triplicate measurements ± SEMs and were analyzed by 1-way analysis of variance (ANOVA) and Bonferroni posttests.