Rainbow Kaposi's Sarcoma-Associated Herpesvirus Revealed Heterogenic Replication with Dynamic Gene Expression

Abstract

Molecular mechanisms of Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named “Rainbow-KSHV,” that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication. IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented.

A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains

“…Technical limitations such as poor sgRNA performance or number may also explain the weak signal from smaller genes such as ORF30 and ORF31, two additional components of the vTA complex. An additional limitation is that less than 25% of cells detectably express the late gene reporter (Figure S1); while this percentage is consistent with previous reports (Brulois et al, 2014;Nakajima et al, 2020), cell models allowing for more efficient late gene expression or detection should further boost the dynamic range and sensitivity. While individual sgRNAs targeting the major transactivator ORF50 behaved as expected (Figure S2b), their positive enrichment in the screen (Figure 1b,c) may represent an unknown role for ORF50 or an artifact of the screen design.…”

A panel of KSHV mutants in the polycistronic kaposin locus for precise analysis of individual protein products

“…We found that kaposin-deficient viruses released similar numbers of DNase-protected viral genomes to WT (Figure 5D). Although this observation suggests that the ability to package genomic material, assemble and release progeny virus does not require the kaposin locus, we and others have observed that extracellular genome copy number does not always correlate with infectious virus (21). To determine whether released virus from each kaposin-deficient latent iSLK cell line were competent to infect naïve cells, we incubated a naïve monolayer of 293T cells with supernatant from reactivated iSLK cells and 24 hours later, stained cells for the latent protein LANA.…”

“…Following reactivation, lytic gene expression follows a prescribed temporal cascade with genome replication marking the transition from early to late gene expression (19, 20). More recent analyses using single cell approaches show that lytic reactivation is quite heterogenous in terms of viral gene expression, host cell responses and outcomes (13, 21–23). The lytic replication phase produces progeny virions enabling transmission of the virus and culminates in cell death.…”

A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains

“…previous reports [45,46], cell models allowing for more efficient late gene expression or detection should further boost the dynamic range and sensitivity. While individual sgRNAs targeting the major transactivator ORF50 demonstrated the expected early-stage block to lytic cycle progression ( Figs 1D, S2D, and S2E), it was surprising that they were positively enriched in the late gene screen (Fig 2B and 2C).…”