Viral microRNA regulation of Akt is necessary for reactivation of Human Cytomegalovirus from latency in CD34+ hematopoietic progenitor cells and humanized mice

Abstract

Human cytomegalovirus (HCMV) actively manipulates cellular signaling pathways to benefit viral replication. Phosphatidyl-inositol 3-kinase (PI3K)/Akt signaling is an important negative regulator of HCMV replication, and during lytic infection the virus utilizes pUL38 to limit Akt phosphorylation and activation. During latency, PI3K/Akt signaling also limits virus replication, but how this is overcome at the time of reactivation is unknown. Virally encoded microRNAs (miRNAs) are a key component of the virus arsenal used to alter signaling during latency and reactivation. In the present study we show that three HCMV miRNAs (miR-UL36, miR-UL112 and miR-UL148D) downregulate Akt expression and attenuate downstream signaling, resulting in the activation of FOXO3a and enhanced internal promoter-driven IE transcription. A virus lacking expression of all three miRNAs is unable to reactivate from latency both in CD34+ hematopoietic progenitor cells and in a humanized mouse model of HCMV infection, however downregulating Akt restores the ability of the mutant virus to replicate. These findings highlight the negative role Akt signaling plays in HCMV replication in lytic and latent infection and how the virus has evolved miRNA-mediated countermeasures to promote successful reactivation. AUTHOR SUMMARYHuman cytomegalovirus (HCMV) infection results in lifelong persistence of the virus through the establishment of latency, and viral reactivation is a significant cause of morbidity and mortality in solid organ and stem cell transplant patients. HCMV latency is established in CD34+ hematopoietic progenitor cells (HPCs) where the virus manipulates cell signaling pathways to maintain the viral genome and remain poised to reinitiate gene expression under the appropriate conditions, although the molecular mechanisms surrounding these processes are poorly understood. HCMV encodes microRNAs (miRNAs) that modulate expression of hundreds of cellular and viral genes and play important roles in regulating signaling in HPCs. In this study, we show that HCMV miR-UL36, miR-UL112, and miR-UL148D coordinately inhibit Akt expression, activation, and downstream signaling through nonconventional mechanisms. A mutant lacking these miRNAs is unable to reactivate from latency, yet complementing Akt regulation restores the ability of the mutant virus to reactivate, pointing to an important role for miRNA-mediated inhibition of Akt to promote HCMV reactivation.