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Published on: November 22, 2011
Human Cytomegalovirus UL78 is a Nuclear-Localized GPCR Necessary for Efficient Reactivation from Latent Infection in
Samuel Medica1, Nicole L Diggins1, Michael Denton1
1Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, Oregon, USA.
Abstract:
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 Gαi 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 Gαi 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 Gαi was the only heterotrimeric Gα 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.
Insights
Human cytomegalovirus (HCMV) UL78 protein is crucial for reactivation from latency in hematopoietic progenitor cells, coupling with Gαi proteins. This viral G protein-coupled receptor is not essential for establishing or maintaining latency.
Area of Science:
- Virology
- Immunology
- Cell Biology
Background:
- Human cytomegalovirus (HCMV) establishes lifelong latency, primarily in myeloid lineage cells.
- Reactivation of latent HCMV poses significant risks, especially for immunocompromised individuals.
- Viral G protein-coupled receptors (GPCRs) like UL78 are implicated in HCMV infection dynamics.
Purpose of the Study:
- To elucidate the role of the HCMV UL78 protein in viral latency and reactivation.
- To investigate the G protein coupling preferences and functional significance of UL78 during reactivation.
- To identify the cellular and viral interactors of UL78 in infected cells.
Main Methods:
- Utilized an in vitro CD34+ hematopoietic progenitor cell (HPC) model for studying HCMV latency and reactivation.
- Generated recombinant HCMV lacking UL78 expression or with mutations in its G protein-coupling motif (DRL).
- Employed Lumit-based assays for G protein coupling, proximity-dependent labeling (TurboID), cell fractionation, immunofluorescence microscopy, and Western blotting.
Main Results:
- Recombinant HCMV lacking UL78 failed to efficiently reactivate from latent CD34+ HPCs.
- UL78 preferentially couples to the Gαi family of G proteins, and this coupling is essential for reactivation.
- Proximity-dependent labeling identified Gαi, trafficking, signaling, and nuclear pore complex proteins in the UL78 interactome, including viral proteins involved in transcription and DNA replication.
- UL78 was confirmed to localize to the nucleus in both fibroblasts and reactivating CD34+ HPCs.
Conclusions:
- HCMV UL78 plays a critical, previously unrecognized role in reactivation from latency in CD34+ HPCs.
- Gαi protein coupling is vital for UL78 function during the reactivation process.
- UL78 interacts with host and viral factors involved in nuclear processes, suggesting a role beyond the cell membrane.
