Abstract
Human Cytomegalovirus (hCMV) infects a broad range of the population and establishes life-long latency in the infected individuals. Periodically the latently infected virus can reactivate and becomes a significant cause of morbidity and mortality in immunocompromised individuals. In latent infection, the viral genome is suppressed in a heterochromatic state and viral gene transcription is silenced. Upon reactivation, the repressive chromatin is remodeled to an active form, allowing viral lytic gene transcription, initiated by the expression of viral Immediate Early (IE) genes. During this process, a number of histone modification enzymes, including histone demethylases (HDMs), play important roles in driving IE expression, but the mechanisms involved are not fully understood. To get a better understanding of these mechanisms, we focused on two HDMs, KDM4 and KDM6, which reverse the repressive histone H3-lysine 9 and lysine 27 methylation, respectively. Our studies show that in lytic infection, both demethylases are important in the activation of viral IE gene expression. Simultaneous disruption of both via genetic or chemical methods leads to severely impaired viral IE gene expression and viral replication. Additionally, in an experimental latency-reactivation model in THP-1 cells, the KDM6 family member JMJD3 is induced upon viral reactivation and its knockdown resulted in reduced IE gene transcription. These findings suggest pharmacological inhibition of these HDMs may potentially block hCMV lytic infection and reactivation, and control the viral infection associated diseases, which are of significant unmet medical needs.
Highlights
HCMV is a beta human herpesvirus with a widely spread infection (50% ~ 80% positive adults)
In the current studies using Human Cytomegalovirus (hCMV) lytic infection and reactivation models, we focused on the regulatory roles of two sets of histone demethylases (HDMs), KDM4 (JMJD2) and KDM6 (UTX/JMJD3), which remove repressive methylation at histone H3K9 and H3K27 respectively
Our results show the following: i) one of the KDM4 members, JMJD2D is involved in activating Immediate Early (IE) transcription, as its knockout by CRISPR led to reduced IE gene expression and yield of infectious progeny; ii) an H3K27 demethylase, JMJD3 of the KDM6 family, plays an important role in CMV lytic infection, as demonstrated by siRNA knockdown studies; iii) there was enhanced repression of viral IE expression and viral yields by the combination of JMJD3 knockdown and JMJD2D knockout; iv) an HDM tool compound ML324 inhibits both KDM4s and KDM6 and potently repressed viral IE gene expression and viral yields; v) JMJD3 is induced upon hCMV reactivation by TPA treatment in THP-1 cells, and its knockdown led to reduced CMV IE induction
Summary
HCMV is a beta human herpesvirus with a widely spread infection (50% ~ 80% positive adults). It is a major cause of disease and death in immunocompromised patients (HIVinfected persons, organ transplant recipients) and is the most significant viral cause of birth. The efficacy of current anti-viral drugs (Ganciclovir & derivative) is compromised by the emergence of drug-resistant viruses [3,4,5,6] and bone marrow toxicity[7], which limits the usefulness of these compounds in tissue transplant patients. There is significant unmet need to develop novel anti-virals to control hCMV infection and the associated diseases
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