Variable Episomal Silencing of a Recombinant Herpesvirus Renders Its Encoded GFP an Unreliable Marker of Infection in Primary Cells

Abstract

The availability of reliable recombinant reporter virus systems has been a great boon to the study of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). Unexpectedly, we found that expression of the ostensibly constitutive green fluorescent protein (GFP) marker was progressively lost during unselected passage in primary rat mesenchymal precursor cells (MM), despite efficient maintenance of latent viral gene expression and episomal partitioning. This repression of EF1-α promoter-driven GFP expression appeared to be passage-dependent, however, since functionally immortalized MM cells derived from long serial passage retained stable expression of GFP following rKSHV.219 infection. Chromatin analysis of cultures that we had infected in parallel demonstrated an increase in repressive H3K27 tri-methylation across the viral episome with the exception of the LANA control region in MM cells infected at early rather than late passage post-isolation. The silencing of GFP expression in the MM cells was reversible in a dose-dependent fashion by the histone deacetylase inhibitor valproic acid, further implicating cellular silencing on incoming viral genomes, and underscoring potential differences in viral gene regulation between primary and functionally immortalized cells. Furthermore, using multispectral imaging flow cytometry, we also determined that the extent of GFP expression per cell among those that were positive did not correlate with the number of LANA dots per nucleus nor the extent of overall LANA expression per cell. This suggests a more complex mode of local gene regulation, rather than one that simply reflects the relative intracellular viral copy number. In sum, we have demonstrated the significant potential for false-negative data when using a constitutive marker gene as a sole means of evaluating herpesviral infection, especially in primary cells.

Targeting the Kaposi’s Sarcoma-associated Herpesvirus Genome With the CRISPR-Cas9 Platform in Latently Infected Cells

“…1). In line with previous reports (26,27), spontaneous loss of GFP expression was evident in cells that were grown in the absence of hygromycin selection, and cells grown in the absence of selection demonstrated a higher degree of GFP loss under all assay conditions as compared with the corresponding cells that were maintained with hygromycin. As expected, targeting of gfp resulted in a dramatic decrease in the fraction of cells expressing GFP, that became more prominent over time.…”

“…Targeting of orf73 generated a significantly larger decline in the quantity of KSHV DNA as compared with gfp (p=0.015) and orf45 (p=0.052) targeting, suggesting that the reduction of KSHV DNA upon targeting of orf73 is due to both incomplete repair and the unique and essential functions of LANA for the maintenance of latent KSHV infection. Loss of KSHV episomes upon targeting of LANA was confirmed by evaluating the number of LANA dots in the nucleus, which was previously shown to correlate with the number of KSHV episomes (26,31). As shown in Fig.…”

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway

“…However, we could not detect significant enrichment of Ago2-2 at the episomal construct in 19T-ROM1 (p ϭ 0.1). Deposition of H3K27Me3 on an episome has been reported recently in Kaposi sarcoma-associated Herpesvirus and linked to gene silencing (48).…”

Epstein–Barr virus enhances genome maintenance of Kaposi sarcoma-associated herpesvirus

“…Overall, BLs were heterogeneous in their response to a dnEBNA-1. In PEL, EBNA-1 and LANA did not colocalize, and the EBV and KSHV plasmids did not commingle, as demonstrated by the (now well-validated) assumption that LANA-dots represent plasmid-chromosome attachment sites (12,13,23,48,61,62). Recent genome structure data are not directly comparable.…”

“…Cell. The number of "LANA dots" in an interphase nucleus correlates with the number of KSHV genomes (48,49). We used 3D immunofluorescence coupled to image reconstruction to count the number of distinct LANA + foci ( Fig.…”