Human Cytomegalovirus UL78 is a Nuclear-Localized GPCR Necessary for Efficient Reactivation from Latent Infection in CD34+ Hematopoietic Progenitor Cells

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that persists throughout the lifetime of the host due in part to the establishment of latency in CD34+ hematopoietic progenitor cells (HPCs) and CD14+ monocytes. HCMV encodes four putative G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78. While the roles of most of these receptors have been investigated, a definitive role for UL78 in HCMV infection has yet to be elucidated. Utilizing an in vitro CD34+ HPC model, we demonstrate that a recombinant virus lacking UL78 protein expression fails to efficiently reactivate from latent infection. Furthermore, we show using a Lumit-based assay that UL78 preferentially couples to the Gi family of G proteins and that a recombinant HCMV containing mutations in the UL78 G protein-coupling DRL motif also fails to reactivate from latent infection. Together our findings indicate that Gi coupling is important for UL78 function during reactivation in latently infected CD34+ HPCs, however the protein is not required to establish or maintain latency. To better understand the role of UL78, we conducted proximity-dependent labeling analyses in HCMV-UL78-TurboID infected fibroblasts and CD34+ HPCs undergoing reactivation from latency. Congruent with our coupling data, we found Gi was the only heterotrimeric G protein in proximity to UL78. Pathway analysis of the UL78 interactome revealed proteins associated with membrane trafficking, signaling, and the nuclear pore complex as enriched in both cell types. In addition, the UL78 interactome contained viral proteins with nuclear localization including viral transcription and DNA replication machinery. Nuclear localization of UL78 was validated using cell fractionation, immunofluorescence microscopy, and proximity-dependent labelling of isolated nuclei. Together, our results provide novel insights into the localization and function of UL78, previously unknown to contribute to reactivation from latent infection. Author SummaryHuman cytomegalovirus (HCMV) remains one of the most widespread viral infections globally. Primary HCMV infection is typically asymptomatic and leads to the establishment of latency in myeloid lineage cells, where the virus persists for the hosts lifetime. Reactivation of latent HCMV can cause severe complications, particularly in immunocompromised individuals such as transplant recipients and people living with HIV. Several factors influence the transition from latent to lytic infection, including signal transduction through the viral G protein-coupled receptors: US27, US28, UL33, and UL78. Using an advanced in vitro model, we show that recombinant viruses lacking UL78 fail to efficiently reactivate from latent infection. Moreover, we show that UL78 preferentially couples to the Gi family of G proteins via a conserved DRL motif, and this coupling is required for efficient reactivation. These results were confirmed by proximity-dependent labeling experiments where we identified Gi and several other proteins involved in trafficking, signaling, transcription, and nuclear localization. Nuclear localization of UL78 was confirmed by cell fractionation, immunofluorescence microscopy, and proximity-dependent labeling in isolated nuclei. Collectively, our results uncover a novel role for UL78 in reactivation from latency and shed new light on its localization and function.