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. 2018 Mar 8;10(3):116.
doi: 10.3390/v10030116.

Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors

Dalibor Miklík  1   2 , Filip Šenigl  3 , Jiří Hejnar  4
Affiliations

Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors

Dalibor Miklík et al. Viruses. .

Abstract

Individual groups of retroviruses and retroviral vectors differ in their integration site preference and interaction with the host genome. Hence, immediately after infection genome-wide distribution of integrated proviruses is non-random. During long-term in vitro or persistent in vivo infection, the genomic position and chromatin environment of the provirus affects its transcriptional activity. Thus, a selection of long-term stably expressed proviruses and elimination of proviruses, which have been gradually silenced by epigenetic mechanisms, helps in the identification of genomic compartments permissive for proviral transcription. We compare here the extent and time course of provirus silencing in single cell clones of the K562 human myeloid lymphoblastoma cell line that have been infected with retroviral reporter vectors derived from avian sarcoma/leukosis virus (ASLV), human immunodeficiency virus type 1 (HIV) and murine leukaemia virus (MLV). While MLV proviruses remain transcriptionally active, ASLV proviruses are prone to rapid silencing. The HIV provirus displays gradual silencing only after an extended time period in culture. The analysis of integration sites of long-term stably expressed proviruses shows a strong bias for some genomic features-especially integration close to the transcription start sites of active transcription units. Furthermore, complex analysis of histone modifications enriched at the site of integration points to the accumulation of proviruses of all three groups in gene regulatory segments, particularly close to the enhancer loci. We conclude that the proximity to active regulatory chromatin segments correlates with stable provirus expression in various retroviral species.

Keywords: gene regulatory elements; genome-wide provirus distribution; provirus silencing; retrovirus integration.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Vectors and the methodological approach used in this study to isolate single-cell clones with stable active proviruses. (A) Schematic representation of retroviral vectors. Avian sarcoma/leukosis virus (ASLV)- and murine leukaemia virus (MLV)-based vectors are of the same structure containing just the respective long terminal repeats (LTR) and enhanced green fluorescent protein (EGFP) for proviral activity detection. The human immunodeficiency virus (HIV)-based vector contains deleted fragments of gag, env and nef genes, full-length tat and EGFP genes and internal ribosomal entry site (IRES). Transcription start sites (TSS) are denoted by broken arrows. ψ, packaging signal. (B) The workflow in obtaining the single-cell clones. K562 cell line was transduced with low multiplicity of infection (MOI) of VSV-G-pseudotyped ASLV-, MLV- or HIV-based vectors. Three days post infection (dpi), Green fluorescent protein positive (GFP+) cells were single-cell sorted to a 96-well plate and cellular clones were established. Cellular clones were examined for the percentage of GFP+ cells 30 and 60 dpi by flow cytometry. Clones containing ≥90% of GFP+ cells were subjected to further examination of proviral integration sites.
Figure 2
Figure 2
Differential expression stability of vectors. (A) The percentage of clones with stable active proviruses (≥90% GFP+ cells) at 30 dpi (blue columns) and 60 dpi (red columns). 100% represents the number of clones obtained after single-cell sorting of GFP+ cells 3 dpi. (B) The percentage of GFP+ cells in single-cell clones analysed at 30 and 60 dpi. Each clone is represented by one blue (30 dpi) and one red (60 dpi) dot at the same position along the x-axis. In most cases, the red dot is lower along the y-axis than the blue dot, which represents the silencing of GFP expression between 30 and 60 dpi. Along the x-axis, clones are ordered by the percentage of GFP+ cells at 30 dpi. The dashed line marks the value of 90% of GFP+ cells. The numbers of clones analysed up to 60 dpi are depicted.
Figure 3
Figure 3
Analysis of genomic features at the integration sites of stable active proviruses. (A) Percentage of proviruses identified inside RefSeq Genes. (B) Orientation of proviruses inside RefSeq Genes relative to the transcription of targeted genes. (C) Distance of proviruses to the nearest TSS of RefSeq Genes (in kb). (D) Distribution of proviruses around TSS. The positive values mark the distance downstream from RefSeq Gene TSS. umMRC, uniquely mapped matched random control.
Figure 4
Figure 4
Transcriptionally active genes targeted by proviral integrations. (A) Percentage of proviruses identified inside RefSeq Genes associated with Tss chromatin state marking active TSS. Dashed whiskers show the original percentages of proviruses inside RefSeq Genes without selection for the presence of the Tss chromatin state. (B) Distance of proviruses to cap analysis of gene expression (CAGE) peaks that mark the site of active TSS. (C) Transcriptional activity of genes with proviruses. Genes were classified into 5 groups according to their mean read per kilobase per million (RPKM) mapped reads. NA marks group with no or very low transcriptional activity. Q1 to Q4 groups contain the genes with RPKM ≥ 1, where Q1 group represents the lowest quartile and Q4 the highest quartile.
Figure 5
Figure 5
Epigenetic features at the integration sites of stably expressed ASLV, HIV and MLV proviruses. (A) Distances of proviruses to the peaks of selected histone modifications that are associated with active promoters and enhancers. (B) Distances of proviruses to merged chromatin segments. Active segments are chromatin segments which are associated with transcriptionally active chromatin. Regulatory segments are chromatin segments associated with active promoters and enhancers. (C) Distances of proviruses to 4 of the 25 chromatin segments analysed, Tss, transcriptional start site segment; Enh, enhancer segment, Elon, elongation segment and Quies, polycomb-repressed segment. The distance is measured as absolute distance to the nearest peak of a histone modification or a chromatin segment. Each dot represents a single provirus. Black dots, real integrations; grey dots, umMRCs.
Figure 6
Figure 6
Comparison of integration sites of stable active proviruses and respective active gene-matched random controls. (A) Statistical p-values of differences between agMRC and integration sites are represented by coloured circles (genes), squares (peaks of histone modifications) and triangles (chromatin segments) and aligned with x axis. agMRCs are created to match the proviral integration sites with the frequency of targeting RefSeq Genes with active TSS part according to the chromatin segment classification. Thin dashed lines mark the p-values of 0.01, 0.05 and 0.1. Values outside the range from 0.01 to 0.1 are located at the lower/upper edge of the chart beyond the dashed lines. Grey symbols represent the p-values of testing integration sites against umMRC. (B) Examples of the charts representing the values of agMRCs and proviral integration sites. From left to right: Tss+RefSeqTSS, absolute distance; Tss+RefSeqTSS, distribution around TSS; peaks of H3K4me1 enrichment; enhancer segments.
Figure 7
Figure 7
The model recapitulating the distribution of stably active proviruses with regard to the genomic and epigenomic features of the integration sites. Yellow arrows, MLV; green, HIV, blue, ASLV.
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