Raf1 promotes successful Human Cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

Abstract

Raf1 is a key player in growth factor receptor signaling, which has been linked to multiple viral infections, including Human Cytomegalovirus (HCMV) infection. Although HCMV remains latent in most individuals, it can cause acute infection in immunocompromised populations such as transplant recipients, neonates, and cancer patients. Current treatments are suboptimal, highlighting the need for novel treatments. Multiple points in the growth factor signaling pathway are important for HCMV infection, but the relationship between HCMV and Raf1, a component of the mitogen-activated protein kinase (MAPK) cascade, is not well understood. The AMP-activated protein kinase (AMPK) is a known regulator of Raf1, and AMPK activity is both induced by infection and important for HCMV replication. Our data indicate that HCMV infection induces AMPK-specific changes in Raf1 phosphorylation, including increasing phosphorylation at Raf1-Ser621, a known AMPK phospho-site, which results in increased binding to the 14-3-3 scaffolding protein, an important aspect of Raf1 activation. Inhibition of Raf1, either pharmacologically or via shRNA or CRISPR-mediated targeting, inhibits viral replication and spread in both fibroblasts and epithelial cells. Collectively, our data indicate that HCMV infection and AMPK activation modulate Raf1 activity, which are important for viral replication. ImportanceGrowth factor signaling plays a critical role in many aspects of viral infection. Here we show that a component of one of these pathways, Raf1, contributes to successful infection of Human Cytomegalovirus (HCMV). We find that AMP-activated protein kinase (AMPK), which is known to be important for HCMV infection, modulates Raf1 phosphorylation throughout infection, and contributes to Raf1 binding to its activating co-factor, 14-3-3. In addition, inhibition of Raf1 inhibits HCMV infection and viral spread. These results suggest a link between two cellular pathways that are important for HCMV replication, AMPK signaling and growth factor receptor signaling, that converge as an important aspect of HCMV infection. This could lead to the potential for new therapeutic targets in immunocompromised individuals afflicted by acute HCMV infection.