✍🏼 Aaron Dirck, Nicole L. Diggins, Lindsey B. Crawford, Wilma D. Perez, Christopher J. Parkins, Hillary H. Struthers, Rebekah Turner, Andrew H. Pham, Jennifer Mitchell, Courtney R. Papen, Daniel Malouli, Meaghan H. Hancock, Patrizia Caposio

🏠 Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, Oregon, USA

📑 Journal of Virology (2023)

Abstract

Human cytomegalovirus (HCMV) is a species-specific virus that establishes a persistent/latent infection in CD34⁺ hematopoietic progenitor cells (HPCs). The ability of HCMV to reactivate from latency is exquisitely linked to changes in cell signaling, which result in HPC differentiation. The Wnt/β-catenin pathway is tightly linked to CD34⁺ HPC homeostasis and differentiation. The majority of the viral and cellular factors involved in the maintenance of HCMV latency and reactivation are still unknown. Our group previously discovered a viral hematopoietic cytokine (pUL7) that promotes cellular differentiation and viral reactivation. Here, we show that the UL7-related RL11 family member UL8 is also required for efficient viral reactivation in CD34⁺ HPCs by interacting with components of the (Wnt)/β-catenin pathway. Pull-down experiments demonstrate that UL8 and β-catenin interact with Dishevelled-2 (DVL2) through their PDZ-binding domains, and this interaction promotes β-catenin stabilization and transcriptional activation. Disrupting the interaction among UL8, β-catenin, and DVL2 blocks HCMV reactivation in vitro and in vivo, similar to deletion of UL8. This is the first observation that Wnt signaling components are essential for HCMV reactivation, unlike α- and γ-herpesvirus that require an active Wnt pathway to maintain latency. Detailed understanding of how the Wnt network is manipulated by herpesviruses will improve our ability to develop targeted therapeutics.

How the WOLF was used in this study
In this study, the WOLF benchtop cell sorter (NanoCellect Biomedical) was used to purify defined populations of HCMV-infected human CD34⁺ hematopoietic progenitor cells (HPCs) for downstream functional assays. Following infection with recombinant HCMV expressing GFP, cells were stained with 7-AAD to exclude non-viable cells and a PE-conjugated anti-human CD34 antibody to identify the target progenitor population. The WOLF sorter enabled gentle, low-pressure fluorescence-based sorting to isolate viable, GFP-positive, CD34⁺ HPCs with high purity. These purified cells were then used in limiting dilution reactivation assays, allowing the authors to accurately quantify viral reactivation frequencies while minimizing cell stress that could confound latency and reactivation outcomes.