FIG. 7.

Recovery of infectious HCMV following in-frame deletion of aa 26 to 33 but not aa 26 to 43 of pp28. (A) Diagram describing the recombinant HCMV BACs derived from wild-type HCMV BAC HB-5. Initially, UL99 deletion created by insertion of a galK cassette to create ΔUL99 BAC, this BAC was repaired with PCR products encoding the deletion/fusion UL99 ORFs, Δ26-33 and Δ26-43. Virus recovery after electroporation of BAC DNA was attempted in two separate experiments, and virus was recovered only from the wild type (WT) and the Δ26-33 mutant on both occasions (day 15 and day 21 postelectroporation) as indicated by the “+” symbol following the stick diagram. The agarose gel to the right demonstrates the size of the UL99 ORF when amplified by oligonucleotides designed to prime from the 5′ and 3′ ends of the ORF. Note the increase in size in the galK cassette containing ΔUL99 BAC and the decrease in size in two individual Δ26-43 BACs. The migration of the PCR product from two individual Δ26-33 BACs appears similar to the product from the wild-type BAC. The predicted amino acid sequence of the PCR products from the nucleotide sequence of the UL99 ORF of the recombinant BACs are shown with the in-frame deletions demarcated. (B) Virus yield curves from HFF cells infected with similar input amounts of WT and Δ26-33 viruses. Note that the growth curves are indistinguishable except for a single point (day 4).