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. 2014 Dec 16;5(6):e02261-14.
doi: 10.1128/mBio.02261-14.

Kaposi's sarcoma-associated herpesvirus genome programming during the early stages of primary infection of peripheral blood mononuclear cells

Hem C Jha  1 , Jie Lu  1 , Subhash C Verma  2 , Shuvomoy Banerjee  1 , Devan Mehta  1 , Erle S Robertson  3
Affiliations

Kaposi's sarcoma-associated herpesvirus genome programming during the early stages of primary infection of peripheral blood mononuclear cells

Hem C Jha et al. mBio. .

Abstract

The early period of Kaposi's sarcoma-associated herpesvirus (KSHV) infection involves the dynamic expression of viral genes, which are temporally and epigenetically regulated. KSHV can effectively infect and persist in endothelial as well as human B cells with different gene expression patterns. To understand the temporal epigenetic changes which occur when KSHV infects the lymphocytic compartment, we infected human peripheral blood mononuclear cells (PBMCs) and comprehensively analyzed the changes which occurred at the binding sites of virally encoded lytic as well as latent proteins along with epigenetic modifications across the KSHV genome during early primary infection. Using chromatin immunoprecipitation (ChIP) assays, we showed that the KSHV genome acquires a uniquely distinct histone modification pattern of methylation (H3K4me3, H3K9me3, and H3K27me3) and acetylation (H3Ac) during de novo infection of human PBMCs. This pattern showed that the epigenetic changes were temporally controlled. The binding profiles of KSHV latent protein LANA and the immediate early proteins RTA and K8 showed specific patterns at different times postinfection, which reflects the gene expression program. Further analysis demonstrated that KSHV can concurrently express lytic and latent genes which were associated with histone modifications at these specific regions on the viral genome. We identified three KSHV genes, K3, ORF49, and ORF64, which exhibited different profiles of histone modifications during the early stages of PBMC infection. These studies established a distinct pattern of epigenetic modification which correlates with viral gene expression temporally regulated during the first 7 days of PBMC infection and provides clues to the regulatory program required for successful infection by KSHV of human PBMCs.

Importance: Kaposi's sarcoma-associated herpesvirus (KSHV) has been documented as one of the major contributors to morbidity and mortality in AIDS patients during the AIDS pandemic. During its life cycle, KSHV undergoes latent and lytic replication. Typically, KSHV maintains a stringent preference for latent infection in the infected B cells. However, 1 to 5% of infected cells undergo spontaneous lytic reactivation. KSHV lytic replication and infection of new cells are likely to be critical for maintaining the population of infected cells which drive virus-associated pathogenesis. Here, we explored the temporal changes of crucial histone marks on the KSHV genome during early infection of human primary peripheral blood mononuclear cells (PBMCs), which are a physiologically relevant system for monitoring primary infection. These results showed that KSHV possessed a distinct pattern of epigenetic marks during early infection of PBMCs. Further, KSHV concurrently expressed lytic and latent genes during this early period. These results now provide new evidence which contributes to understanding the molecular mechanism that regulates viral gene expression during early infection.

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Figures

FIG 1
FIG 1
Global patterns of histone modification across KSHV genome during early primary infection. The histone modification ChIP assays were performed with wt KSHV-infected PBMCs at 1, 2, 4, and 7 days p.i. using the antibodies for histone H3K4me3, H3K9me3, H3K27me3, and H3Ac. ChIP DNA was assayed by quantitative PCR using a genome-wide array of primers across the KSHV genome. Approximate KSHV genome positions are indicated on the top of the panel. IE, immediate early; dpi, days postinfection.
FIG 2
FIG 2
Analysis of KSHV latency and lytic life cycle during early primary infection at 1, 2, 4, and 7 days p.i. ChIP assays were performed with wt KSHV-infected human PBMCs using the antibody for KSHV latent protein LANA and lytic proteins RTA and K8. ChIP DNAs were assayed by quantitative PCR using a genome-wide array of primers across the KSHV genome. Approximate KSHV genome positions are written on the top of the panel. IE, immediate early; dpi, days postinfection.
FIG 3
FIG 3
Schematic representation of the experimental setup for genome-wide ChIP analysis during KSHV early primary infection. (A) Experimental flow schematic for ChIP assay during KSHV early primary infection. (B) A representative picture of sonicated DNA samples from KSHV-infected PMBCs at 1, 2, 4, and 7 days p.i. After sonication, 10 µl sonicated DNA samples was run on a 1.5% agarose gel to verify that sonication had resulted in ~500-bp DNA fragments. Numbers at left are sizes in bp. (C) Analysis of histone modification and gene regulation of K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, and cells were harvested at 1, 2, 4, and 7 days p.i. ChIP assays were performed with wt KSHV-infected human PBMCs using the antibodies for histone modification markers (H3K4me3, H3K9me3, H3K37me3, and H3Ac) and KSHV proteins (LANA, RTA, and K8). Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed on a StepOnePlus real-time PCR system. dpi, days postinfection.
FIG 4
FIG 4
Analysis of mRNA levels for K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, RTA1st, RTAall, and LANAp, and cells were harvested at 1, 2, 4, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of K3 (A), ORF49 (B), and ORF64 (C) were quantified by qRT-PCR on a StepOnePlus real-time PCR system. (D) The mRNA levels of K3, ORF49, and ORF64 in BJAB, BCBL1, and BC3 cells were analyzed by qRT-PCR. dpi, days postinfection; RQ, relative quantity.
FIG 5
FIG 5
Methylated-CpG island recovery assay (MIRA) utilizes the high enrichment of the MBD2B/MBD3L1 (2B/3L) complex for double-stranded methylated DNA (61). It is able to recover the methylated DNA without the use of bisulfite conversion or antibody recognition and is sensitive enough to detect low-density methylation of a single methylated CpG nucleotide. Analysis of active CpG levels for promoters of K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. (A) The CpG islands within K3, ORF49, and ORF64 promoters were confirmed by Meth Primer software. (B) Relative degree of methylation in KSHV episome from BCBL1 cells examined by MIRA approach. We validated the MIRA using primers across the KSHV K3, ORF49, and ORF64 promoters. (C and D) Human PBMCs were infected by wt KSHV, RTA1st, RTAall, and LANAp, and cells were harvested at 1, 2, 4, and 7 days p.i. Samples were sonicated and subjected to MIRA, which utilized the high enrichment of the MBD2B/MBD3L1 complex for double-stranded methylated DNA. The activities of CpG islands within the K3, ORF49, and ORF64 promoters were determined by qRT-PCR. WT, wild type; dpi, days postinfection; FD, fold change in antibody compared to IgG control; RE, relative enrichment.
FIG 6
FIG 6
Bivalent histone modification patterns at K3, ORF49, and ORF64 promoters. Sequential ChIP assays were performed with antibodies directed against H3K4me3 and H3K27me3 during the first and second rounds of immunoprecipitation (IP). (A) K3 promoter. (B) ORF49 promoter. (C) ORF64 promoter. FD, fold change in antibody compared to IgG control.
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