Fig 1.
BAC16 K8.1pr-mIFP2 infected iSLK cells enables fluorescent readout of late gene activity.
a) The late gene reporter is expressed 48–96 hours post-reactivation. iSLK cells infected with either the wildtype BAC16 or the late-gene reporter modified BAC16 were reactivated, and reporter activity was monitored every day for 96 hours. b) Sensitivity of late gene reporter to inhibitors of viral DNA replication phosphonoacetic acid (PAA) and cidofovir (CDV). Reporter activity was monitored by flow cytometry 72 hours after reactivation. Inhibitors of viral DNA replication restricted reporter activity. c) Virion production of late gene reporter virus after transfer of supernatant to uninfected HEK293T cells. 72 hours post-reactivation, viral supernatant was filtered and transferred to naïve HEK293T cells; infection of HEK293T cells was monitored by expression of the BAC16-encoded, constitutive EGFP. Error bars are standard error from three technical replicates. d) Virion production after supernatant transfer to uninfected HEK293T cells. SAFE denotes sgRNAs targeting the host in regions without expected function [47]. Non-coding viral 1 and 2 indicates sgRNAs targeting two viral regions free of ORFs. Each bar represents an independent sgRNA, and error bars represent standard error from three independent reactivations.
Fig 2.
Pooled tiling screen of KSHV genome for modifiers of late gene expression.
a) Design of viral tiling screen for late gene expression. iSLK cells were infected with KSHV encoding a far-red reporter of late-gene activity. Cells were lentivirally infected with Cas9-blast and subsequently the KSHV tiling sgRNA library. After 1–2 weeks of latency to allow for editing, cells were reactivated into the lytic cycle by doxycycline and sodium butyrate. 48 hours post reactivation, cells were trypsinized, fixed, and sorted for high and low late-gene expression. The sgRNA locus in each population was then sequenced to calculate enrichment. b,c) To calculate significance, sgRNAs targeting each annotated region of the genome were grouped and a signed log Mann-Whitney p-value was calculated comparing each viral region to the negative control sgRNAs using an average enrichment from two replicates. Viral ORFs sorted by significance (b) or genome location (c). A positive log p-value indicates this region promotes late gene expression when disrupted, and a negative log p-value indicates this region is required for expression of late genes. d,e) Three independent sgRNAs from the screen targeting each indicated ORF were individually cloned and delivered to late-gene reporter iSLK cells. Virion production was measured by supernatant transfer to uninfected HEK293T cells (d) and late-gene reporter activity (e). Error bars are standard error from three independent reactivations. Activated, unreactivated, and parent samples treated with the viral DNA replication inhibitor cidofovir (CDV) were included as controls, along with three vSAFE guides targeting an unexpressed region of the viral BAC. P values were calculated using a single-tailed, equal variance Student’s t-test, with the least significant value used when compared to each individual vSAFE sgRNA. NS indicates non-significance (P>0.01).
Fig 3.
Deep viral sequencing identifies ORF46 requirement for viral DNA replication.
a) Design of targeted deep-sequencing experiment. Four viral loci were amplified and deep-sequenced: ORF21, ORF18, ORF34, and ORF46. b) Median enrichment across the coding region of the gene of out-of-frame indels in replicating cells relative to latent cells (Viral DNA replication) or the supernatant relative to latent cells (Virion production). Error bars are standard error from two replicates. One replicate of ORF34 supernatant sample was excluded due to uneven coverage. c,d,e) Three individual sgRNAs were lentivirally delivered to KSHV-infected iSLK cells targeting the indicated viral ORF. Error bars are standard error from four independent reactivations. CDV-treated, reactivated, and unactivated parental cells are included as controls, along with three vSAFE sgRNAs targeting an ORF-free region of the BAC. P values were calculated using a single-tailed, equal variance Student’s t-test, with the least significant value used when compared to each individual vSAFE sgRNA. NS indicates non-significance (P>0.01). c) Reactivation and early gene expression was measured using a KSHV virus containing a HaloTag fusion to a viral early gene, ORF68. d) Measuring viral DNA replication by percentage positive EdU staining. A 2-hour pulse of EdU was delivered 48 hours post reactivation. Cells were then fixed, and click chemistry was used to measure EdU incorporation by flow cytometry. Unreactivated cells were used to establish gates for flow cytometry, where subgenomic EdU incorporation was measured as viral DNA replication. e) DNA was extracted 48 hours post reactivation, and qPCR of the viral promoter of ORF57 was used to measure the amount of viral DNA.
Fig 4.
Targeted deep sequencing reveals essential domains of ORF46.
a,b) Smoothed signal from in-frame mutations across the a) ORF18 and b) ORF34 loci in comparison between replicating and supernatant genomes. Functional domain labeling for ORF18 [39] and ORF34 [40]. c) Smoothed signal from in-frame mutations across the ORF46 loci in comparison between latent and replicating genomes. In-frame mutations were defined as insertions or deletions whose size where divisible by three. The number of mutations was pooled in a 100 bp window and enrichment was calculated relative to the enrichment of out-of-frame mutations. A negative value indicates that in-frame mutations were relatively depleted at the indicated region. Previously described domains and residues are indicated in red if they appear depleted and blue if they do not. d) vDNA replication was measured for iSLK cells infected with KSHV encoding either wildtype ORF46, a catalytic domain mutant of ORF46 (Q87L, D88N), or a DNA-binding domain mutant of ORF46 (H210L). Cells were reactivated, and after 48 hours DNA was extracted, and viral DNA quantity was measured by qPCR of the viral ORF57 promoter. Quantity is relative to unreactivated samples. Error bars are standard error from four independent replicates.