The ViHiHippo project aimed to uncover how human cytomegalovirus (HCMV) proteins, specifically the viral G protein-coupled receptors (vGPCRs) US28 and UL78, affect the Hippo signaling pathway, which is important for controlling cell growth and organ size. The project was divided into three main objectives:
Objective 1: Identify and Characterize US28 Signaling
To understand how US28 affects the Hippo pathway, we used HEK293 cells, which are commonly used in lab experiments. These cells were modified to produce US28 and a reporter that shows activity in the Hippo pathway. We used gene-editing techniques and various inhibitors to identify which proteins are involved in US28's effects.
Key achievements:
• Discovered that US28's constant activity (without external signals) drives changes in the Hippo pathway, while activation by chemokines (signaling molecules) does not.
• Found that US28 signals through a specific group of proteins called the Gq family to influence the Hippo pathway.
• Determined that other proteins, including G12/13, beta-arrestins, and PLCβ1-4 enzymes, are not necessary for US28's effects on the Hippo pathway.
• Confirmed that a protein called YAP is essential for US28 to affect the Hippo pathway.
Objective 2: Investigate UL78 Signaling
Unlike US28, UL78 does not use the usual signaling proteins. We created various modified versions of UL78 to find out which parts of the protein are important for its function. We also used gene-editing and inhibitors to study its signaling.
Key achievements:
• Found that a specific part of UL78, called the DRY motif, is not needed for its signaling, but a cluster of serine and threonine amino acids at the end of the protein is crucial.
• Determined that the usual signaling proteins, including G proteins and beta-arrestins, are not involved in UL78's effects on the Hippo pathway.
• Developed nanobodies (small antibody fragments) that bind to UL78 and can change its activity.
Objective 3: Characterize Effects in Cancer
To see how our findings apply to a more realistic setting, we studied the effects of US28 and UL78 in U251 glioblastoma cells, a type of brain cancer cell. We used systems that allow us to control the production of US28 and UL78 in these cells and measured the activity of the Hippo pathway.
Key achievements:
• Confirmed that US28 affects the Hippo pathway in U251 glioblastoma cells.
• Found that US28 increases the expression of a marker called CD133, which is associated with cancer stem cells, in U251 cells.
• Observed that US28-expressing U251 cells show changes in shape that indicate a lack of differentiation (maturation into specialized cells).
• Found evidence that US28 may promote stem cell-like properties in glioblastoma cells, including increased CD133 expression and lack of differentiation.
• Obtained inconclusive results for UL78's effects in U251 cells, possibly due to low levels of UL78 production.
Overall, the ViHiHippo project provided important insights into how HCMV proteins US28 and UL78 affect the Hippo signaling pathway and their potential roles in cancer progression. These findings could help develop new treatments for HCMV-associated cancers.
