Human Cytomegalovirus UL7, miR-US5-1, and miR-UL112-3p Inactivation of FOXO3a Protects CD34 ⁺ Hematopoietic Progenitor Cells from Apoptosis

Abstract

Human cytomegalovirus (HCMV) infection of myeloid lineage cells, such as CD34+ hematopoietic progenitor cells (HPCs) or monocytes, results in the upregulation of antiapoptotic cellular proteins that protect the newly infected cells from programmed cell death. The mechanisms used by HCMV to regulate proapoptotic cellular proteins upon infection of CD34+ HPCs have not been fully explored. Here, we show that HCMV utilizes pUL7, a secreted protein that signals through the FLT3 receptor, and miR-US5-1 and miR-UL112-3p to reduce the abundance and activity of the proapoptotic transcription factor FOXO3a at early times after infection of CD34+ HPCs. Regulation of FOXO3a by pUL7, miR-US5-1, and miR-UL112 results in reduced expression of the proapoptotic BCL2L11 transcript and protection of CD34+ HPCs from virus-induced apoptosis. These data highlight the importance of both viral proteins and microRNAs (miRNAs) in protecting CD34+ HPCs from apoptosis at early times postinfection, allowing for the establishment of latency and maintenance of viral genome-containing cells. IMPORTANCE Human cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals and is a significant problem during transplantation. The virus can establish a latent infection in CD34+ hematopoietic progenitor cells (HPCs) and periodically reactivate to cause disease in the absence of an intact immune system. What viral gene products are required for successful establishment of latency is still not fully understood. Here, we show that both a viral protein and viral miRNAs are required to prevent apoptosis after infection of CD34+ HPCs. HCMV pUL7 and miRNAs miR-US5-1 and miR-UL112-3p act to limit the expression and activation of the transcription factor FOXO3a, which in turn reduces expression of proapoptotic gene BCL2L11 and prevents virus-induced apoptosis in CD34+ HPCs.

Regulation of Latency and Reactivation by Human Cytomegalovirus miRNAs

“…Additional studies performed in the absence of infection or using cell lines that are not permissive for HCMV latency have suggested that HCMV miRNAs can inhibit apoptosis through suppression of SLC25A6/ANT3 [75] and immediate early gene X-1 (IEX1) [76]. More recently, HCMV miR-US5-1 and miR-UL112-3p were shown to target FOXO3a [77], a member of the mammalian Forkhead Box O family of transcription factors that promotes mitochondrial-dependent and -independent mechanisms of apoptosis induction [78,79]. FOXO3a binds to the promoters of pro-apoptotic regulators such as Bcl-2-like protein 11 (Bim) and stimulates its expression [80].…”

“…The activity of FOXO3a is regulated by PI3K/AKT and MEK/ERK signaling, which mediate phosphorylation and translocation of FOXO3a to the cytoplasm [81][82][83]. The FOXO3a transcript is downregulated by HCMV miR-US5-1 and miR-UL112-3p and the protein is targeted for phosphorylation and inactivation by the HCMV FLT3L homolog UL7 [77]. Both the miRNAs and UL7 are expressed at early times post-infection of CD34 + HPC [15,77] and reduce FOXO3a levels and activity to limit the induction of apoptosis [77] in this cell type ( Figure 2).…”

“…The FOXO3a transcript is downregulated by HCMV miR-US5-1 and miR-UL112-3p and the protein is targeted for phosphorylation and inactivation by the HCMV FLT3L homolog UL7 [77]. Both the miRNAs and UL7 are expressed at early times post-infection of CD34 + HPC [15,77] and reduce FOXO3a levels and activity to limit the induction of apoptosis [77] in this cell type ( Figure 2). This study demonstrates a coordination between an HCMV protein and HCMV miRNAs to promote survival of infected cells in a way that supports the establishment of HCMV latency.…”

“…In addition, a cellular miRNA family expressed in CD34 + HPCs targets HCMV IE86 to prevent lytic replication [84]. Additionally, miR-US5-1 and miR-UL112 act synergistically with HCMV US7 to downregulate Forkhead Box O3 (FOXO3a) and thereby prevent apoptosis during latency establishment [77]. HCMV proteins are shown in green and HCMV miRNAs are shown in red.…”

“…Inhibits apoptosis during latency establishment via FOXO3a downregulation [77] Targets HCMV US7 [68] Inhibits production of IL-6 and RANTES through IKKα and IKKβ targeting [100] Restructures the endocytic recycling compartment to limit cytokine secretion [102] Targets FADD, may affect apoptosis signaling [74] miR-US5-2…”