Reactivation of latent Human Cytomegalovirus (HCMV) in CD34(+) hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14(+) macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34(+) HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34(+) HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34(+) HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34(+) HPCs, indicating that expression of EGR-1 inhibits viral reactivation. Since EGR-1 promotes CD34(+) HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.

Non-coding RNAs (ncRNAs) play essential roles in multiple aspects of the life cycles of herpesviruses and contribute to lifelong persistence of herpesviruses within their respective hosts. In this chapter, we discuss the types of ncRNAs produced by the different herpesvirus families during infection, some of the cellular ncRNAs manipulated by these viruses, and the overall contributions of ncRNAs to the viral life cycle, influence on the host environment, and pathogenesis.

Non-coding RNAs (ncRNAs) play essential roles in multiple aspects of the life cycles of herpesviruses and contribute to lifelong persistence of herpesviruses within their respective hosts. In this chapter, we discuss the types of ncRNAs produced by the different herpesvirus families during infection, some of the cellular ncRNAs manipulated by these viruses, and the overall contributions of ncRNAs to the viral life cycle, influence on the host environment, and pathogenesis.

Emerging evidence indicates that human cytomegalovirus (HCMV) manipulates host cell signaling pathways using both proteins and noncoding RNAs. Several studies have shown that HCMV induces NF-kappa B signaling early in infection, resulting in the induction of antiviral proinflammatory cytokines with a subsequent reduction of these cytokines late in infection. The mechanism for late cytokine reduction is unknown. In this study, we show that HCMV microRNAs (miRNAs) miR-US5-1 and miR-UL112-3p target the I kappa B kinase (IKK) complex components IKK alpha and IKK beta to limit production of proinflammatory cytokines in response to interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha). Transfection of miR-UL112-3p and miR-US5-1 mimics reduced endogenous IKK alpha and IKK beta protein levels, and site-directed mutagenesis of the 3' untranslated regions (UTRs) identified the binding sites for each miRNA. Infection with mutant viruses lacking these miRNAs resulted in increased levels of IKK alpha and IKK beta proteins, an impaired ability to control NF-kappa B signaling at late times of lytic infection, and increased production of proinflammatory cytokines compared to wild-type virus in cell types relevant to HCMV infection in vivo. These phenotypes were rescued by preexpression of miR-US5-1 and miR-UL112-3p in infected cells or by a miR-US5-1/miR-UL112-3p double mutant virus that expresses short hairpin RNAs (shRNAs) targeting IKK alpha and IKK beta, demonstrating the gene specificity of the miRNAs. These observations describe a mechanism through which HCMV miRNAs expressed late in the infectious cycle downregulate proinflammatory cytokine production to create a cellular proviral environment.IMPORTANCE Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in transplant recipients and causes hearing loss and mental retardation when acquired congenitally. Initial events during HCMV infection result in the activation of NF-kappa B signaling, which culminates in the production of IL-6, CCL5, and TNF-alpha. Several viruses have developed mechanisms to block the antiviral effects of these cytokines. We show here that two HCMV miRNAs, miR-US5-1 and miR-UL112-3p, specifically downregulate IKK alpha and IKK beta signaling factors necessary to propagate NF-kappa B signaling and subsequent IL-6, CCL5, and TNF-alpha production. Regulation of these proinflammatory cytokines during lytic infection and during latency is critical to viral survival in the host.