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. 2019 Dec 17;9:19236. doi: 10.1038/s41598-019-55508-w

Figure 3.

Figure 3

Genetic stability of pp71 deficient HCMV TR3. (A) Stable expression of HIVgag upon multiple passages. MRC-5 cells were mock-infected or infected with TR3ΔUL78gag or TR3ΔUL82gag at an MOI of 0.01 and the supernatant was harvested at full CPE. For sequential passages (p1-p20), TR3ΔUL82gag in the supernatant was titered on BJ-pp71 cells and used for the next round of infection at MOI = 0.01. Lysates from cells harvested at the indicated passages were electrophoretically separated and probed for expression of the shown viral proteins or cellular actin by immunoblot. The blots for each protein are shown as cropped from different parts of the same gel. (B) SNP analysis of TR3ΔUL82gag upon serial passaging. Viral DNA was harvested at the indicated passages from the same samples as in (A) and subjected to Next Generation Sequencing (NGS) on a MiSeq platform. The position and frequency of SNPs are shown along the viral genome. Two SNPs were enriched upon passaging: T-C at position 59972 and C-T at position 82959 resulting in an Asn-Asp change in UL45 and an Asp-Asn change in UL55. (C) SNP analysis of TR3 upon serial passaging. TR3 was serially passaged as in (A) and viral DNA was harvested at the indicated passages and subjected to NGS. The position and frequency of SNPs are shown along the viral genome. One SNP was enriched upon passaging: C-A at position 82489 resulting in a Ser-Ile change in UL55.