Oral Presentation ESA-SRB-AOTA 2019

Placental ex vivo model infection for pathogenesis studies of Congenital Cytomegalovirus  (#87)

William Rawlinson 1 2 3 4
  1. Serology and Virology Division, NSW Health Pathology Microbiology, Prince of Wales Hospital, Sydney, NSW, Australia
  2. School of Women’s and Children’s Health, University of New South Wales, Sydney, NSW, Australia
  3. School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia
  4. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia

Congenital CMV infection (cCMV) affects ~350 Australian infants annually. It is extremely challenging clinically, particularly with lack of vaccine or effective, licensed pharmaceutical treatments, combined with practical and ethical problems studying fetal infection. Infection may cause fetal malformation and in severe cases fetal and neonatal death. Fetal injury may be caused directly by fetal infection with cell damage or indirectly by placental infection, inflammation in response to infection and fetal undernutrition..  

We study how CMV dysregulates trophoblast function, at protein and molecular levels. Our studies are of viral-cellular protein interactions implicated in cCMV pathogenesis including i) cyclin-mediated complex interactions with viral proteins includinge vCDK pUL97 complexes, ii) DYRK (Dual specificity tyrosine kinases, protein family important in neuronal development) and CDK (cell cycle regulators) interactions with CMV proteins, iii) CMV interactions with dysregulation of the cell protein Wnt (a signalling pathway important in placental development).

We study these events using ex vivo first trimester TEV-1 trophoblast cells and ex vivo placental explants infected with laboratory-adapted (AD169) or genetically-intact (Merlin) CMV strains, or naturally infected clinical placentae. Infection causes accumulation and relocalisation of DYRK1A proteins to cell cytoplasm and cytoplasmic virion assembly complexes, with sequestration of DYRK1B to nuclear replication compartments. Western blots show this accumulation  results from upregulated DYRK1A and DYRK1B protein expression, resulting from transcriptional upregulation. Treatment of TEV-1 and placental explants with novel DYRK inhibitors significantly inhibits HCMV replication, indicating these cellular kinases are essential during HCMV placental replication.

We also show CMV alters expression of receptor tyrosine kinase ROR2 to modulate Wnt5a-stimulated trophoblast migration in the non-canonical pathway. CMV infection increases expression of Wnt-binding receptor ROR2. Ectopic ROR2 expression reduces T-cell-specific (TCF)/lymphoid enhancer-binding factor (LEF)-medicated transcription and inhibits Wnt5a-induced trophoblast migration. Downregulation of ROR2 using siRNA duplexes rescues CMV-induced reduction in trophoblast migration. These data suggest CMV alters cell protein functions, with implications for placental development and function.