In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome
Fig 5
A) Comparing in vitro growth kinetics of FL-RhCMV/Rh13.1/apt in fibroblasts to 68–1 and 68–1.2 RhCMV in a multistep growth curve. Primary rhesus fibroblasts were infected with 68–1, 68–1.2 and FL-RhCMV/Rh13.1/apt at an MOI of 0.01 on day 0. Cell culture supernatants were harvested on the indicated days and virus titers were determined by TCID50. Two biological repeats of each sample were titrated in duplicate and the arithmetic mean of the results were graphed. B) FL-RhCMVΔRh13.1gag, depicted above the figure, shows increased infection of epithelial cells compared to 68–1 RhCMV. Primary rhesus fibroblasts or rhesus retinal epithelial cells (RPE) were infected with MOIs of 0.3 or 10, respectively, and all experiments were performed in triplicates. After 48 hours post infection, cells were harvested, fixed, permeabilized, stained with a RhCMV specific antibody [62] and analyzed by flow cytometry. Statistical significance was shown using an unpaired t-test with a p-value significance threshold of <0.05. C) Synthetic N-terminal peptide (Peptide 1) of OR14I1 blocks PRC-positive RhCMV infection of RPE cells. Virus inoculum of 68–1, 68–1.2 or FL-RhCMVΔRh13.1gag was preincubated with peptide 1 (32.6uM) or DMSO before infection of RPE cells (MOI 4.0). On day 3 pi, cells were fixed, permeabilized, immunostained for IE2 to identify infected cells, stained for DNA (blue), and imaged (4×). Results were then quantified and plotted. Data represent the mean of n = 3 experiments ±SD. Statistical significance was determined using an unpaired t-test. D) Rh159, the RhCMV homolog of UL148, is upregulated in FL-RhCMV/Rh13.1/apt. Relative mRNA copy numbers of Rh159 (UL148) and Rh137 (UL99) were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using specific probes. The data shown represent the mean of triplicate repeats (+/- SEM). Unpaired student t-tests with a p-value significance threshold of <0.05 were performed to show statistical significance in both graphs comparing FL-RhCMV/Rh13.1/apt to either 68–1 RhCMV or 68–1.2 RhCMV at 48 hpi.