Mechanisms of Human Cytomegalovirus Latency

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DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) establishes lifelong latent infections in a host and reactivates with devastating consequences within immunosuppressed patients, underscoring the need to identify the conditions that dictate HCMV latency. To this end, we have developed and reported a novel HCMV latency model system utilizing Kasumi 3 cells (K3s) that recapitulates ALL the key stages of HCMV reactivation allowing us to experimentally test conditions critical for HCMV latency that were previously not possible. This proposal aims to test the hypothesis that the antagonistic relationship between miRNA-induced suppression of HCMV IE genes and cytokine-mediated induction of IE genes is a major determinant of the switch from latent to lytic infection. We base this concept on our exciting observations that cellular encoded hsa-miR-200 family members target the essential HCMV Immediate Early (IE) 2 protein. Additionally, we show that IE2 can induce viral Early (E) promoters within the context of the genome and independently of additional viral proteins, providing the first confirmation that IE2 is a key regulator of HCMV viral lytic transcription. In addition, we show that inflammatory cytokines potently initiate lytic reactivation of HCMV in our K3 cels that can be blocked by treatment with an NF-kB inhibitor. Based on these observations we favor a model (derived from published observations with Herpes Simplex Virus) wherein latency is established by chromatization of the viral genome resulting in suppression of viral transcription. However, an added level of suppression mediated by miRNA targeting of IE transcripts would suppress viral translation assuring latency maintenance. We offer the novel concept that HCMV has co-opted cellular miRNAs to additionally restrict viral reactivation. We seek to extend these studies on the mechanisms underlying HCMV latency and reactivation. In Aim 1, we will determine the biological role of cellular encoded miRNAs on restricting HCMV IE translation as well as evaluate the requirement of viral encoded miRNAs on HCMV latency. Aim 2 will address the mechanisms by which inflammatory cytokines function to reactivate HCMV as well as define the requirements of cellular transcription factors on promoting the latent to lytic switch. Completion of the aims above will contribute significantly to our knowledge on the mechanisms of viral latency and lytic reactivation. This work will lay the foundation for identifyig novel therapeutic targets for a virus that is a significant global pathogen.