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. 2017 Jun 26;91(14):e00437-17.
doi: 10.1128/JVI.00437-17. Print 2017 Jul 15.

Cell Culture Systems To Study Human Herpesvirus 6A/B Chromosomal Integration

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Free PMC article

Cell Culture Systems To Study Human Herpesvirus 6A/B Chromosomal Integration

Annie Gravel et al. J Virol. .
Free PMC article

Abstract

Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5 h), cells were processed for single-cell cloning and analyzed for chromosomally integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using quantitative PCR (qPCR), droplet digital PCR, and fluorescent in situ hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase-positive (HeLa, MCF-7, HCT-116, and HEK293T) and telomerase-negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes, including U39, U90, and U100, without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression, as well as the release of infectious HHV-6A/B from the integrated state.IMPORTANCE The analysis and understanding of HHV-6A/B genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the present study, we describe two quantitative cell culture viral integration systems. These systems can be used to define cellular and viral factors that play a role in HHV-6A/B integration. Furthermore, these systems will allow us to decipher the conditions resulting in virus gene expression and excision of the integrated viral genome resulting in reactivation.

Keywords: HHV-6; chromosomal integration; ddPCR; telomere.

Figures

FIG 1
FIG 1
Characterization of clones with integrated HHV-6. (A and B) DNA samples from U2OS and a U2OS-BP6 clone containing ciHHV-6B were analyzed by ddPCR. After PCR, the content of droplets was analyzed for the presence of HHV-6 (blue), cellular gene RPP30 (green), HHV-6+ RPP30 (orange), or no DNA (gray). (C) The average number of HHV-6 copies/cell from U2OS clones with ciHHV-6A and ciHHV-6B was determined by ddPCR. DNA from cell clones without HHV-6 integration and DNA samples from known iciHHV-6A+/B+ subjects were used as negative and positive controls (neg and pos ctl), respectively. (D) FISH analysis of a U2OS clone containing ciHHV-6A. Telomeres (TMR) and HHV-6A were visualized using Cy3-labeled telomeric probe (red) and HHV-6A using Alexa-488 (green)-labeled probe. An enlarged portion of the image is presented on the right.
FIG 2
FIG 2
Maintenance and growth of cells containing ciHHV-6 over time. (A) U2OS cells were infected with HHV-6A or HHV-6B and cultured for 150 days. At various times, DNA was extracted and analyzed by ddPCR for estimation of ciHHV-6 frequency. (B) U2OS and U2OS-AP3 cells (containing ciHHV-6A) were analyzed for their growth rates. Cells were plated on day 0, and on days 3 and 7 the number of cells was determined. Results are expressed as mean cell number ± standard deviations (SD) from triplicate cultures.
FIG 3
FIG 3
Spontaneous HHV-6 gene expression from ciHHV-6A/B U2OS clones. (A) Eleven U2OS clones containing ciHHV-6A were analyzed for the spontaneous expression of IE and U100 by RT-qPCR. Results are expressed as mean ratios ± SD of viral genes relative to GAPDH mRNA levels. (B) U2OS-AP3 (ciHHV-6A+) and U2OS-BP6 (ciHHV-6B+) were analyzed for the spontaneous expression of IE1 protein by IFA using purified anti-IE1 rabbit IgG coupled to Alexa 488. Nuclei were stained blue using 4′,6-diamidino-2-phenylindole (DAPI).
FIG 4
FIG 4
Spontaneous and induced HHV-6 gene expression from ciHHV-6A+/B+ U2OS and MCF-7 clones. (A to F) The expression of HHV-6A/B U90, U39, and U100 genes from ciHHV-6A+ (AP3 and WTR-BAC#25) and ciHHV-6B+ (BP6) U2OS clones was determined by RT-qPCR at the indicated times following TNT (TPA, NaBy, TSA, and hydrocortisone) or SAHA stimulation. Results are shown as mean fold induction ± SD relative to untreated (resting) cells. (G to I) Expression of U90, U39, and U100 from ciHHV-6A+ (MCF7-66A) and ciHHV-6B+ (MCF-7 32B) MCF-7 clones was determined by RT-qPCR at the indicated times following TNT or SAHA stimulation. Results are shown as mean fold induction ± SD relative to untreated (resting) cells. NS, not stimulated. (J) GFP expression in resting and TSA-activated U2OS clone 9 containing integrated WTR-BAC HHV-6A. Results are representative of a minimum of three independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01.
FIG 5
FIG 5
Generation of HHV-6A and HHV-6B doubly integrated U2OS cells. U2OS-AP3 (ciHHV-6A+) and U2OS-BP6 (ciHHV-6B+) were superinfected with HHV-6B and HHV-6A, respectively, and submitted to single-cell cloning. Clones positive for two viruses were identified by FISH and ddPCR. Metaphase spreads (A and C) and interphase nuclei (B and D) of AP3#19 (A and B) and BP6#48 (C and D) showing the presence of two distinct HHV-6 hybridization signals (arrows). (E and G) Droplet digital PCR analysis of cell lines containing integrated HHV-6A and integrated HHV-6B. (E) ciHHV-6A+ and ciHHV-6B+ cell lines assayed together to produce a droplet plot with multiple clusters. Gray droplets are negative for template. Blue droplets are positive for HHV-6A. Orange droplets are positive for HHV-6B. Green droplets are positive for RPP30. (F) ddPCR analysis of the U2OS AP3 superinfected with HHV-6B to generate clone 19 containing both HHV-6 species. On average, this clone contained 2.9 HHV-6A and 2.2 HHV-6B genomes/cell. (G) ddPCR analysis of the U2OS BP6 clone superinfected with HHV-6A, resulting in clone 48 containing both HHV-6 species. On average, this clone contained 0.6 HHV-6A and 0.6 HHV-6B genomes/cell.

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