Human Herpesvirus 6B

Abstract Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after c irradiation in both permissive and nonpermissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following c irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein

Direct Repeat 6 from Human Herpesvirus-6B Encodes a Nuclear Protein that Forms a Complex with the Viral DNA Processivity Factor p41

The SalI-L fragment from human herpesvirus 6A (HHV-6A) encodes a protein DR7 that has been reported to produce fibrosarcomas when injected into nude mice, to transform NIH3T3 cells, and to interact with and inhibit the function of p53. The homologous gene in HHV-6B is dr6. Since p53 is deregulated in both HHV-6A and -6B, we characterized the expression of dr6 mRNA and the localization of the translated protein during HHV-6B infection of HCT116 cells. Expression of mRNA from dr6 was inhibited by cycloheximide and partly by phosphonoacetic acid, a known characteristic of herpesvirus early/late genes. DR6 could be detected as a nuclear protein at 24 hpi and accumulated to high levels at 48 and 72 hpi. DR6 located in dots resembling viral replication compartments. Furthermore, a novel interaction between DR6 and the viral DNA processivity factor, p41, could be detected by confocal microscopy and by co-immunoprecipitation analysis. In contrast, DR6 and p53 were found at distinct subcellular locations. Together, our data imply a novel function of DR6 during HHV-6B replication

Complex formation of DR6(6B) and p41 during HHV-6B infection.

<p>(A) Western blotting of input lysate before immunoprecipitation. Probed with anti-DR6. (B) Western blotting of p41 and control IgG immunoprecipitation with or without HHV-6B-infection at 48 hpi. Probed with anti-DR6. (C) Western blotting of lysates after one round of p41 and IgG immunoprecipitation. Blots were probed with anti-DR6. A representative of two experiments is shown.</p

Expression of <i>dr6</i> mRNA and protein in HHV-6B-infected cells.

<p>(A–D) Relative expression of <i>dr6</i> mRNA using <i>tbp</i> mRNA as reference. HCT116 (A and B) or MOLT3 (C and D) cells were incubated in the presence or absence of CHX (A and C) or PAA (B and D) and lysed at the indicated timepoint followed by mRNA extraction. (E) Anti-DR6 polyclonal antibodies identified a 44 kDa band by Western blotting, which was blocked by the addition of a DR6(6B) peptide. The peptide sequence was as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007457#pone-0007457-g001" target="_blank">Figure 1D</a>. (F) Western blotting of HCT116 cell extract at 24, 48, and 72 hpi. Expression of DR6(6B) and GAPDH is shown. A representative of 3 experiments is shown.</p

Association of DR6(6B) and the p41 viral DNA processivity factor.

<p>(A) Confocal laser scanning microscopy of DR6(6B) (green) and p41 (red) of HCT116 cells at 48 hpi. DNA was stained with DAPI (blue). Columns with DR6/p41 and DNA/DR6/p41 indicate an overlay of the individual stainings. (B) High magnification of DR6/p41 overlay. (C) Co-localization coefficient computed by the LSM710 software. Ch1-DR6 indicates the fraction of DR6(6B) that co-localized with p41. Ch2-p41 indicates the fraction of p41 that co-localized with DR6(6B). Values are the average of measurements on 14 cells with an indication of standard deviation on top of the bars. A representative of three experiments is shown.</p

Alignments of <i>dr7(6A)</i>, <i>dr6(6A)</i>, <i>orf-1(6A)</i>, and <i>dr6(6B)</i>.

<p>(A) Schematic alignment of the <i>dr6</i> gene from HHV-6A (NC_001664, nt 4725-6720) with the <i>Sal</i>I-L fragment (X73675), the <i>orf-1</i> from the <i>Sal</i>I-L fragment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007457#pone.0007457-Thompson1" target="_blank">[14]</a>, the <i>dr6(6A)</i> and <i>dr7(6A)</i> open reading frame <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007457#pone.0007457-Gompels1" target="_blank">[9]</a>. (B) Schematic alignment of <i>dr6</i> gene of HHV-6B (NC_000898, nt 5027-7203). Arrows indicate the primers used for amplification by real-time PCR. (C) Alignment of the 3′-end of <i>dr7(6A)</i>, <i>dr6(6A)</i>, <i>orf-1(6A)</i>, and <i>dr6(6B)</i>. Nt 6664 indicates the position where a frameshift takes place in <i>dr6(6A)</i> and <i>dr7(6A)</i>. (D) Alignment of the C-terminal tail of DR7(6A), DR6(6A), ORF-1, and DR6(6B). The amino acid sequence for the antigenic peptide is indicated.</p

Nuclear distribution of DR6(6B) in a pattern similar to TBP.

<p>(A) Separation of HCT116 lysate at 48 hpi into nuclear (NE) and cytoplasmic (CY) fractions. Western blots were probed with anti-DR6, anti-7C7 to indicate HHV-6B infection, anti-RCC1 as a nuclear protein control, and anti-GAPDH as a cytoplasmic control. (B) Confocal laser scanning microscopy of localization of DR6 and TBP at 24, 48, and 72 hpi. DNA was stained with DAPI. Columns with DR6/TBP and DNA/DR6/TBP indicate an overlay of the individual stainings. A representative of three experiments is shown.</p

HHV-6B infection rescues cells from p53-dependent apoptosis.

<p>(<b>A & B</b>) Western blot analysis of PARP cleavage in cells either mock-treated or infected with HHV-6B for 48 hrs. The cells were subsequently treated with mock, leupeptin (10 µM), UV radiation (10 min exposure followed by 4 hrs of incubation), doxorubicin (0.2 µg/ml for 24 hrs), γ radiation (30 Gy followed by 24 hrs of incubation), or MG132 (10 µM). 7C7 was used as infection control and GAPDH was used as loading control. (<b>C & D</b>) Western blot analyses with antibodies against PARP, GAPDH, and 7C7 (infection control) on lysates from HCT116 (C) or MOLT3 (D) cells treated with varying doses of UV radiation (10, 20, 240, 600 J/cm² followed by 4 hrs of incubation). (<b>E</b>) ATP cell viability assay on MOLT3 cells with or without HHV-6B infection and treated with varying amounts of UV (0, 2.5, 5, 10, 15, 20, 40 and 60 J/cm² followed by 4 hrs of incubation) or γ radiation (0 and 30 Gy followed by 24 hrs of incubation). (<b>F</b>) Western blot analyses with antibodies against PARP, p53, PUMA, p21 or GAPDH (loading control) on lysates from HCT116 cells either mock-treated or infected with HHV-6B for 24 hrs followed by γ radiation (30 Gy) and 24 hrs of incubation. (<b>G</b>) Confocal microscopy images of HCT116 cells either mock-treated or infected with HHV-6B for 24 hrs, γ-irradiated (30 Gy) followed by 24 hrs of incubation. The cells were analyzed for cleaved caspase-3 (Alexa 488 green), DR6 (Alexa 546 red), and DAPI (blue).</p

HHV-6B infection leads to nuclear accumulation of phosphorylated p53.

<p>(<b>A</b>) Western blot analyses of HCT116 cells infected for 24, 48, or 72 hrs and analyzed with antibodies against p53 p-Ser15, p53 p-Ser46, p53, 7C7 (infection control), or GAPDH (loading control). (<b>B</b>) Western blot analyses of nuclear (NF) and cytoplasmic (CF) fractions from mock-treated or HHV-6B-infected HCT116 cells (48 hpi). The membranes were stained with antibodies against p53, p53 p-Ser46, 7C7 (infection control), RCC1 (nuclear control), or GAPDH (cytoplasmic control). (<b>C</b>) Western blot analyses of HCT116 cells mock-treated or HHV-6B-infected for 24 hrs followed by γ irradiation (30 Gy) and additional incubation for 24 hrs. The membrane was probed with antibodies against p53, p53 p-Ser15, or GAPDH (loading control). Numbers at the bottom of the figure indicate fold induction of Ser15 phosphorylation relative to GAPDH. (<b>D & E</b>) Confocal microscopy on MOLT3 cells infected with HHV-6B for 24 hrs followed by staining with antibodies against the viral protein p41 (green), p53 (red) DAPI (blue). The p53 antibodies were either the monoclonal DO-7 (D) or a polyclonal antibody (FL393) (E). Arrows point to nuclear p53 staining. (<b>F</b>) Confocal microscopy on MOLT3 cells infected with HHV-6B for 24 hrs followed by staining with antibodies against p53 phospho-Ser20 (green), 7C7 (red) and DAPI (blue). (<b>G</b>) Confocal microscopy on MOLT3 cells infected with HHV-6B for 24 hrs, γ-irradiated (30 Gy) for 24 hrs followed by staining with antibodies against the viral protein DR6 (green), p53 (DO-7) (red) DAPI (blue).</p

U19 expression inhibits p53-dependent check-point arrest.

<p>(<b>A</b>) Cell cycle analyses of wt HCT116 cells, HCT116 cells stably expressing U19, and HCT116-p53^−/− cells. The cells were stained with NIM-DAPI and analyzed by flow cytometry. Four independent experiments are shown. (<b>B</b>) Quantification of the G1, S and G2 distributions, using the Dean-Jett-Fox algorithm in the Flow-Jo software program. An average of the four independent experiments from (A) is shown. Error bars indicate SD. The p values in G1 are: 1) p = 0.0005; 2) p = 0.0003; and 3) p = 0.49 (p values were determined using a t-test). (<b>C</b>) Analysis of p21 promoter activation using a p21-pro-<i>luciferase</i> construct. HCT116 wt cells and HCT116 cells stably expressing U19 were transfected with a p21-Luc promoter construct, γ-irradiated (30 Gy followed by 24 hrs of incubation) and analyzed for luciferase induction. Y-axis depicts relative light units (RLU). A representative out of two experiments is shown. Values are average of triplicate measurements with error bars indicating SD. (<b>D</b>) Real-time PCR on mRNA from HCT116 wt, U19S and p53^−/− cells with <i>MDM2</i> or <i>TBP</i> specific primers. <i>MDM2</i> mRNA levels are represented relative to <i>TBP</i>. Measurements were performed in duplicate. Two independent experiments is shown.</p