Productive Kaposi Sarcoma Associated Herpesvirus (KSHV) infection involves translating, folding, and modifying abundant viral proteins in the Endoplasmic Reticulum (ER). The heightened demand for ER function can disrupt ER homeostasis and activate the Unfolded Protein Response (UPR), a collection of stress-signaling pathways in charge of restoring ER homeostasis. Three ER-resident sensors/transducers, the kinase/RNase IRE1, the kinase PERK, and the membrane-bound transcription factor ATF6, govern the UPR. Their activation either promotes adaptation or causes apoptosis if homeostasis is not restored. A master regulator of the UPR and ER function is the HSP70 ER-resident molecular chaperone GRP78/BiP. This abundant chaperone modulates the activation of the UPR sensors and promotes protein folding in the ER lumen to restore homeostasis. Typically, BiP expression is transcriptionally upregulated in response to ER stress by ATF6. However, our results show that lytic reactivation of KSHV in iSLK-219 cells leads to the ATF6-independent accumulation of GRP78/BiP. The upregulation of GRP78/BiP is critical during the lytic cycle as pharmacological inhibition of genetic depletion of GRP78/BiP reduces viral gene expression and viral particle production. Moreover, our results suggest that BiP activity promotes the survival and growth of KSHV-infected B-cells and lymphatic endothelial cells, and that KSHV-infected cells are uniquely sensitive to a BiP inhibitor, HA15. Inhibition of BiP also leads to the reduction in replication of other DNA viruses, including the beta-herpesvirus human cytomegalovirus (HCMV), the alpha-herpesvirus herpes simplex virus 1 (HSV-1), and the poxvirus vaccinia virus (VV), indicating that the pro-viral role for BiP is not exclusive for KSHV. Our results highlight BiP as a promising target for developing broad-spectrum antivirals and potential therapies for treating KSHV-related malignancies.

ABSTRACT

Inhibition of the eIF4E-kinase MNK as a Strategy for Controlling Kaposi’s Sarcoma-associated Herpesvirus Replication

by

Catya-Luth Faeldonea

During viral infection, viruses hijack the host protein translation machinery to efficiently produce their own proteins for subsequent infection. Cap-dependent translation relies on the formation of the heterotrimeric cap-binding complex, eIF4F. The formation and activity of this complex is regulated by different pathways that modulate the phosphorylation status of the eIF4F components. Two such pathways, p38 and ERK, converge on the phosphorylation of the MAP kinase interacting kinases 1 and 2 (Mnk1 and Mnk2), which are the sole kinases of the cap-binding protein eIF4E, an integral component of eIF4F. These kinases are required for tumor formation and in response to cellular and environmental stress, but are negligible for normal cellular functions and development. The role of these kinases during viral infection have been thoroughly investigated. In the context of infection by herpesviruses, several studies demonstrated that MNK activity is upregulated in cells infected by Herpes Simplex Virus-1 (HSV-1), Human Cytomegalovirus (HCMV) and Kaposi’s Sarcoma Associated Herpesvirus (KSHV). The phosphorylation of eIF4E is critical for herpesvirus replication as genetic and pharmacological inhibition reduces virus production. My goal is to exploit MNK1/2 and eIF4E phosphorylation as a potential therapeutic target for KSHV.KSHV is an oncogenic virus and the etiological agent for Kaposi’s Sarcoma (KS), Multicentric Castleman’s Disease, and primary effusion lymphoma (PEL), which are diseases commonly diagnosed in immunocompromised patients. KSHV is prevalent in 50% of the population in sub-Saharan Africa. Unfortunately, despite its continued occurrence, there are currently no FDA-approved therapeutics against KSHV-related diseases. Because of this, there is strong interest in finding new targets for therapeutic intervention. The eIF4E kinase MNK1/2 has been considered a promising target to reduce KSHV replication. Previous studies demonstrate that inhibition of MNK1 with the small molecule inhibitor CGP57380 (CGP) reduced viral replication; however, this compound does not have an effect on the second eIF4E kinase, MNK2, and has since been reported to have numerous off-target effects. To better evaluate the potential of MNK1/2 as therapeutic targets for KSHV infection, I evaluated pharmacological inhibition of both MNK isoforms with the compound Tomivosertib, and genetic ablation of MNK1/2 in KSHV infected cells. My studies show that treatment with Tomivosertib downregulates the levels of the KSHV structural proteins and inhibits viral replication. Using CRISPRi technology, I established a MNK1/2 double knockdown, KSHV-infected cell line to evaluate the effect of genetic ablation of MNK1/2 on KSHV infection. As seen with the Tomivosertib treated cells, viral reactivation was reduced by over 50% in these cells. These findings suggest that MNK1 and MNK2 are promising therapeutic targets for the treatment of KSHV and its associated malignancies.

Zika virus (ZIKV), a mosquito-borne flavivirus, causes severe congenital eye disease. However, the molecular mechanisms underlying retinal pathologies are largely unknown in human modeling. This dissertation explores the mechanisms of viral pathogenesis in the retina using stem cell-derived retinal organoids (ROs) as a model for Zika virus (ZIKV) and other retinal-targeting herpes simplex virus 1 (HSV1) infections. Utilizing immature ROs, we elucidate specific impacts of early trimester ZIKV infections on retinal progenitor and ganglion cells, demonstrating significant disruptions to organoid growth and health. Our findings underscore the critical role of the AXL receptor in mediating retinal ZIKV infection, suggesting its potential as a therapeutic target. Conversely, modeling with mature ROs reveals the rapid and destructive pathogenesis of HSV1, highlighting the vulnerability of photoreceptor and Müller glial cells to adult retinal necrotizing infections.Further, through single-cell RNA sequencing (scRNA-seq), we reveal distinct cellular responses to ZIKV and HSV1, including unique gene expression patterns associated with cellular stress responses and interferon signaling. Specifically, our work details the modulation of the unfolded protein response (UPR) by ZIKV infection and identifies key pharmacological targets for inhibiting viral replication and enhancing cell viability. Additionally, we demonstrate the potent antiviral effects of interferon-alpha and beta in ZIKV-infected ROs, emphasizing the importance of bystander immune stimulation. Our findings provide a nuanced understanding of retinal viral infections, offering novel insights into the highly complex cellular and molecular underpinnings of ZIKV pathogenesis in the retina and underscoring the value of retinal organoids as a model system for studying infectious diseases.

Background

Results

Conclusions