Kaposi's Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter.

Endrit Elbasani,Adam Grundhoff,Jere Weltner,Thomas Günther,Veijo Nurminen,Päivi M Ojala,Diego Balboa,Silvia Gramolelli,Francesca Falasco,Timo Otonkoski

doi:10.3390/v12090952

Abstract

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus–host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.

Highlights

Kaposi’s sarcoma-associated herpesvirus (KSHV) has been etiologically linked to three malignancies, namely Kaposi sarcoma (KS), primary effusion lymphoma (PEL), the plasmablastic variant of multicentric Castleman disease (MCD) and to two systemic inflammatory diseases: KSHV-inflammatory cytokine syndrome (KICS) and KS immune reconstitution syndrome (KS-IRIS) [1,2,3,4,5]
We set out to investigate whether a repurposed deficient Cas9 (dCas9) fused to transcription activators can be guided to the KSHV genome to induce the expression of ORF50 and thereby trigger the complete
destabilization domain (DD)-dCas9-VP192 has been previously used to induce the expression of multiple genes simultaneously to control cell differentiation [21,22]

Summary

Introduction

Kaposi’s sarcoma-associated herpesvirus (KSHV) has been etiologically linked to three malignancies, namely Kaposi sarcoma (KS), primary effusion lymphoma (PEL), the plasmablastic variant of multicentric Castleman disease (MCD) and to two systemic inflammatory diseases: KSHV-inflammatory cytokine syndrome (KICS) and KS immune reconstitution syndrome (KS-IRIS) [1,2,3,4,5]. While PEL is a B cell tumor in which the virus is found predominantly in a latent form, KSHV lytic reactivation is involved in the pathogenesis of the other KSHV-related diseases [6,7,8,9]. KSHV lytic cycle is initiated by the expression of the replication and transcription activator (RTA). It has been demonstrated that ORF50 expression is necessary and sufficient to trigger the complete lytic replication cycle which culminates with the production and release of infectious viral progeny [9,10]. As for instance, in lymphatic endothelial cells KSHV undergoes asynchronous and spontaneous lytic reactivation in a subset of infected cells resulting in the production of high titers of infectious virus [11,12]

Methods

Results

Conclusion