Fig 5.

Generation of recombinant viruses. (A) Schematic diagram of UL94 mutations incorporated into the viral genome. Nucleotide changes incorporated to change cysteine residues to alanine are indicated. AA, amino acid. (B) UL94 mutant virus stocks were generated from HAUL94-complementing HFF cells. Viruses were then used to infect noncomplementing HFF cells at a multiplicity of 0.01 PFU/cell, and total virus was harvested at 21 days postinfection. Titers of progeny virus were determined on HAUL94-complementing HFF cells. (C) HFF cells were infected at a multiplicity of 0.05 PFU/cell, and total protein was harvested at 120 h postinfection. Levels of viral proteins were analyzed by Western blotting. IE1/2, immediate-early proteins 1 and 2. (D) UL99 mutant BACs were transfected into HFF cells, and virus replication was monitored by cytopathic effect and GFP expression from the viral genome. Recovery of infectious virus from BAC-transfected cells for each mutant is indicated. Rep, repair.