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. 2020 Aug 27;12(9):952.
doi: 10.3390/v12090952.

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

Endrit Elbasani  1 , Francesca Falasco  1 , Silvia Gramolelli  1 , Veijo Nurminen  1 , Thomas Günther  2 , Jere Weltner  3 , Diego Balboa  3 , Adam Grundhoff  2 , Timo Otonkoski  3 , Päivi M Ojala  1   4
Affiliations

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

Endrit Elbasani et al. Viruses. .

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.

Keywords: CRISPRa; DD-dCas9-VP192; KSHV; KSHV lytic cycle; KSHV reactivation; Kaposi’s sarcoma-associated herpesvirus; ORF50; RTA; dCas9.

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Conflict of interest statement

We declare no conflict of interest.

Figures

Figure 1
Figure 1
Targeting a nuclease-deficient Cas9 (dCas9) based activator to the ORF50 promoter induces Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation. (A) Schematic representation of destabilizing domain (DD)-dCas9-VP16 activation domain (VP192) stabilization upon addition of trimethoprim (TMP). (DHFR: DD–dihydrofolate reductase-derived destabilization domain, VP192–12X VP16) (B) DD-dCas9-VP192 localization in HEK293-DD-dCas9-VP192_rKSHV.219 treated with DMSO or TMP (500 nM) for 72 h. Intracellular localization of DD-Cas9-VP192 was detected by anti-HA antibody staining and nuclei were counterstained with Hoechst 33342. Representative images (left) and quantification of DD-Cas9-VP192 fluorescence signal (violin plot, right) in the area occupied by each individual nucleus (n > 500, Student’s t test, *** −p < 0.001). Below the violin plot, immunoblot analysis for the indicated proteins of HEK293-DD-dCas9-VP192-rKSHV.219 cells treated with DMSO or TMP (500 nM) for 72 h. Actin normalized band quantification is shown for DD-dCas9-VP192, below the bands. (C) Upper panel: schematic representation of DD-dCas9-VP192 binding sites upstream of ORF50 upon expression of the indicated single-guide RNAs (sgRNAs). Numbers indicate nucleotides upstream of ORF50 start codon (indicated as 0). Lower panels: GFP and RFP expression in HEK293_DD-dCas9-VP192_rKSHV.219 non-transfected (NT) or transfected with vectors expressing the indicated sgRNA and treated with TMP (500 nM) for 72 h. RFP is a marker of the rKSHV.219 lytic replication cycle. (D) Quantification of ORF50, ORF57, ORF45 and K8.1 mRNA levels in cells treated as in (C). Each dot represents a biological replicate, bars show average, error bars show SD. (E) Quantification of rKSHV.219 titers in the supernatant of HEK293-DD-dCas9-VP192-rKSHV.219 cells transfected with sg50-2-expressing plasmid and treated with DMSO or TMP (500 nM) for 72 h. Each dot represents a biological replicate, bars show average, error bars show SD, Student’s t test, *** −p < 0.001.
Figure 2
Figure 2
Activators fused to dCas9 can trigger the expression of ORF57 without disrupting the viral latency. (A) A schematic representation of DD-dCas9-VP192 binding sites upstream of the ORF57gene upon expression of the indicated sgRNAs. Numbers indicate nucleotides upstream of ORF57 start codon (ATG), shown as 0. (B–G) HEK293_DD-dCas9-VP192_rKSHV.219 cells were transfected with vectors expressing the indicated sgRNA or left non-transfected (NT) and treated with TMP (500 nM) for 72 h. (B) Analysis of ORF57 mRNA levels. (C) Analysis of ORF50 and ORF57 mRNA levels. (D) Immunoblot analysis for the indicated proteins. (E) Quantification of the intracellular KSHV genome copies. (F) Quantification of rKSHV.219 titers in the supernatant. (G) Heatmap of KSHV gene expression after RNA-seq analysis of samples in (C) (n = 2 biological replicates). The read counts were normalized to NT samples and to genome copy numbers calculated in (E). Dark red tiles show values above 50. (B,C,E,F) Each dot represents a biological replicate, bars show average, error bars show SD.
Figure 3
Figure 3
Simultaneous targeting of the dCas9-activator at two sites on the ORF50 promoter enhances KSHV reactivation. (A–C) HEK293_DD-dCas9-VP192_rKSHV.219 cells were transfected with vectors expressing the sgRNA sg50-2, sg50-3, both (sg50-2+3) or left non-transfected (NT) and treated with TMP (500 nM) for 72 h. (A) Quantification of ORF50, ORF57, ORF45 and K8.1 mRNA levels. (B) Immunoblot analysis for the indicated proteins. (C) Quantification of rKSHV.219 titers in the supernatant. (A and C) Each dot represents a biological replicate, bars show average, error bars show SD. One-Way ANOVA followed by Dunnett’s correction for multiple comparisons testing were used to calculate whether differences between sg50-2, sg50-3 and sg50-2+3 groups were significant (*** −p < 0.001, ** −p < 0.01, * −p < 0.05).
Figure 4
Figure 4
dCas9-activator is more efficient than ectopic expression of ORF50 to induce the viral lytic replication. (A–D) HEK293_DD-dCas9-VP192_rKSHV.219 cells were transfected with vectors expressing the sgRNA sg50-2 and sg50-3 (sg50-2+3) or Myc-ORF50 and treated with TMP (500 nM) for 72 h. (A) Representative images showing GFP (upper row) and RFP (lower row) expression of cells transfected with sg50-2 and sg50-3 (sg50-2+3), Myc-ORF50 or non-transfected (NT). RFP is a marker of the rKSHV.219 lytic replication cycle. (B) Immunoblot analysis for the indicated proteins. (C) Quantification of rKSHV.219 titers in the supernatant. (D) Quantification of ORF50 expression after staining of the cells with an anti-ORF50 antibody. Nuclei were counterstained with Hoechst 33342. (C,D) In the violin plot, n > 450 nuclei/condition. In the bar graphs, each dot represents a biological replicate, bars show average and error bars show SD. Student’s t test was used to calculate whether differences between Myc-ORF50 and sg50-2+3 groups were significant (** −p < 0.01, *** −p < 0.001).
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