Transcriptome profiling of human cytomegalovirus-infected trophoblasts

Placental infection plays a central role in the pathogenesis of congenital human cytomegalovirus (HCMV) infections and is a cause of fetal growth restriction and pregnancy loss. HCMV can replicate in some trophoblast cell types, but it remains unclear how the virus evades antiviral immunity in the placenta and how infection compromises placental development and function. Human trophoblast stem cells (TSCs) can be differentiated into extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STBs). This study assessed the utility of TSCs as a model of HCMV infection in the first trimester placenta. TSCs and TSC-derived EVTs and STBs were infected with HCMV (TB40/Ewt-mCherry). RNA was isolated from infected cells at 24, 48, and 72 hours post-infection and Illumina RNA-Sequencing was used to measure viral and host gene expression. Viral gene expression in TSCs does not follow the kinetic patterns observed during lytic infection in fibroblasts. Canonical antiviral responses were largely not observed in HCMV-infected TSCs and TSC-derived trophoblasts. Rather, infection dysregulated factors involved in cell identity, differentiation, and WNT signaling. CT27 and CT29 TSCs were grown in self-renewal media or differentiated into EVT and STBs for 3 days. Cells were infected with HCMV (TB40/Ewt-mCherry) at a MOI of 1 (2 replicates per cell type and time point). RNA was extracted from mock- and CMV-infected cells at 24, 48, and 72 hours post-infection. Infected TSCs were flow sorted based on mCherry flourescense into three populations: mock-infected, infected cells that expressed high levels of mCherry, and infected cells that expressed low levels of mCherry. RNA was extracted from these cells and Illumina sequencing libraries were prepared using the SMARTer Pico Mammalian V2 kit (Takara) and sequenced using and Illumina NovaSeq (S4 Flow Cell, 2x150-bp PE reads). RNA was extracted directly from EVTs and STBs and used to prepare TruSeq Stranded mRNA (Illumina) sequencing libraries. These samples were sequenced using and Illumina NovaSeq (S4 Flow Cell, 2x150-bp PE reads).