The experimental setup:

<p>1. Total RNA was extracted. 2. Reverse transcription was performed in two different setups i) using random hexamers to collect all possible types of RNA transcripts and ii) using oligo dT primers to gain polyadenylated types of RNA transcripts. 3) Real-time PCR with primers designed to specifically recognize N- and C-terminus of β-galactosidase cDNA was used to amplify sequences at both 5′-end (PCR-1) and 3′-end (PCR-2) of β-galactosidase RNA transcripts.</p

Inhibition of expression from extrachromosomal HIV promoter by PCI.

<p>A β-galactosidase reporter assay was used to measure expression from the promoter within the pHIV-lacZ plasmid following transfection into a Tat-expressing cell line (H1299-Tat). Results were compared with expression from Tat-expressing cell line with pHIV-lacZ stably integrated into the cellular genome (H1299-HIV). The requirement for Tat to promote expression from HIV promoter was illustrated in H1299 (Tat-negative) control cells transiently transfected with pHIV-lacZ. The Y axis represents relative β-galactosidase activity and the error bars illustrate the standard deviation of three independent biological replicates.</p

OCII and Rosc suppress expression from extrachromosomal genetic elements.

<p>(A) MCF-7 cells transfected with pSV-lacZ were treated either with OCII (8 µM) or Rosc (20 µM). Relative levels of β-galactosidase activity were determined in a chromogenic (ONPG) enzyme assay. The Y axis represent<b>s</b> relative β-galactosidase activity and the error bars illustrate the standard deviation of three independent biological replicates. (B) OCII and Rosc induce wild type p53-mediated transactivation of p21^WAF−1. MCF-7 (wild type p53) cell line was treated either with OCII (8 µM) or Rosc (20 µM) for 24 h and analyzed for the expression of p53, p21^WAF−1 and actin by immunoblotting. The numbers represent the fold changes of samples to untreated controls.</p

The effect of PCIs on the integrity of synthesized RNA.

<p>We compared amounts of total and full length polyadenylated transcripts of β-galactosidase gene after treatments with OLII and Rosc. We quantified total and full length transcripts of β-galactosidase gene in cell lines H1299-HIV (B) and H1299-Tat transiently transfected by pHIV-LacZ (A) by using qRT-PCR. PCIs stimulated the RNA synthesis from viral promoters ((B) H1299-HIV PCR-1, Random hexamers (A) H1299-Tat, PCR-1, Random hexamers) however viral promoters incorporated in the cellular genome generated higher amounts of full length RNA transcripts ((B) H1299-HIV, PCR-2, OLIGO dT). The amount of full length polyadenylated transcripts produced from extrachomosomal viral promoters after treatments with PCIs were significantly lower ((A) H1299-Tat, PCR-2, OLIGO dT). Data were determined by relative quantification with control PCR-1 and control PCR-2 as the calibrators. The Y axes represent the fold change of β-galactosidase RNA transcripts after PCIs treatment to the amount of β-galactosidase RNA transcripts in controls. The error bars illustrate the standard deviation of three independent biological replicates.</p

The effect of OCII and Rosc on phosphorylation of RNA polymerase II CTD.

<p>(A) Phosphorylation of RNA polymerase II CTD in wild type p53 cells either with stably integrated SV40 (COS-1) or SV40-negative cells (CV-1) was determined. The phosphorylation of both Ser 2 and Ser 5 was inhibited after 12 h treatment either with 8 µM OCII or 20 µM Rosc in all tested cell lines. (B) Comparison of RNA polymerase II CTD phosphorylation in cells expressing Tat with pHIV-lacZ stably integrated into cellular genome (H1299-HIV) or with pHIV-lacZ as a part of extrachromosomal DNA (H1299-Tat). The phosphorylation of Ser 2 and Ser 5 in both cell lines was inhibited after PCI treatment for 12 h. The numbers represent the fold changes of samples to untreated controls.</p

OCII and Rosc suppress T-antigen expression.

<p>(A) OCII and Rosc suppress T-antigen expression and restore p53–mediated transactivation of p21^WAF−1 during SV40 infection. MCF-7 (wild type p53) cell line infected with SV40 was treated either with 8 µM OCII or 20 µM Rosc, either immediately or 16 h post-infection. (OCII 16 h, Rosc 16 h). Infected cells were harvested at 45 h and then analyzed for the expression of large T-antigen, p53, p21^WAF−1 and actin by immunoblotting. (B) Inhibition of expression from viral promoter integrated into cellular genome. Cell lines CV-1 and COS-1 were treated either with 8 µM OCII or 20 µM Rosc and analyzed for the expression of large T-antigen, p53, p21^WAF−1 and actin by immunoblotting. CV-1 cell line was used as SV40-negative control cell line (no large T-antigen expression). The numbers represent the fold changes of samples to untreated controls.</p

Olomoucine II differs from roscovitine only by an additional hydroxyl group on 6-benzyl group.

<p>Olomoucine II differs from roscovitine only by an additional hydroxyl group on 6-benzyl group.</p

PMP comparing wild-type and US2-deficient HCMV shows a requirement for US2 in the downregulation of cell surface targets.

<p>(A) Scatter plots of proteins identified in PMP and quantified by 2 or more unique peptides. Fold change (HFF infected with wild-type HCMV versus HFF infected with ΔUS2 HCMV) is shown as log₂ ratio on the x axis and the summed peptide intensity on the y-axis as log₁₀. Proteins unaltered by HCMV US2 gene deletion locate to the center of the plots (0-fold change), whereas proteins left or right of center represent respectively proteins down- or up-regulated by HCMV in a US2-dependent manner. Significance B was used to estimate p values. (<b>B</b>) US2 is required for integrin and thrombomodulin downregulation during HCMV infection. HFF cells infected with GFP tagged HCMV ΔUL16-18 or ΔUL16-18ΔUS1-11 (moi 0.5) were analyzed by cytofluorometric analysis of the indicated proteins at 72 hours post-infection. (<b>C</b>) HFFs infected with HCMV wild-type or ΔUS2 (moi 0.5) were analyzed by flow cytometry at 72 hours post-infection. Cell surface staining for MHC-I enabled gating for HCMV infected (MHC-I^lo) cells (left panel) and subsequent analysis of the integrin and thrombomodulin expression (right panels). See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004811#ppat.1004811.s007" target="_blank">S3 Table</a></b>.</p

UL141 retains CD112 in the ER and enhances US2 mediated degradation.

<p>ER retention of CD112 is mediated by UL141 and only detected upon depletion of US2 or TRC8. (<b>A</b>) HFF infected with mock, HCMV wild-type, ΔUS2 or ΔUL141 (moi 5) were analyzed for the indicated proteins at 72 hours post infection, in the presence (shTRC8+) or absence (shTRC8-) of TRC8 depletion. (<b>B</b>) UL141 retains CD112 in the ER prior to TRC8-dependent degradation by US2. THP-1 cells stably expressing US2, US11, UL141-Myc or shTRC8 were solubilized in 1% digitonin, immunoprecipitated with anti-Myc antibody and the indicated proteins visualized by immunoblot. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004811#ppat.1004811.s004" target="_blank">S4 Fig</a></b>.</p

US2 induces the proteasomal degradation of integrin α4 and α5 and prevents maturation of the β1 integrin.

<p>(<b>A and B</b>) US2 induces proteasomal degradation of integrins α4 and α5. THP-1 cells stably expressing US2, US11 or depleted of TRC8 (shTRC8) were analyzed by immunoblots for the indicated proteins. Cells were treated with the proteasome inhibitor lactacystin for 18 hours before harvest as indicated. (<b>C-D</b>) Integrin 4 is proteasomally degraded in US2-expressing cells. THP-1 cells expressing integrin α4-HA were labeled with [³⁵S]methionine-cysteine and pulse-chased for the indicated times in the presence or absence of MG132, followed by integrin α4 immunoprecipitation using the HA-tag <b>(E)</b> β1 integrin accumulates in its precursor form in the presence of US2. THP-1 cells were pulse-chase labelled as above and endogenous β1 integrin was immune precipitated using anti-integrin β1 antibody. (<b>F and G</b>) US2 associates with integrin α4 and recruits the TRC8 E3 ligase via its cytosolic tail. Cells stably expressing full-length US2 (US2) or US2 with Δ186–199 residues (US2ΔC') in combination with TRC8-HA or integrin α4-HA were solubilised in 1% digitonin, immunoprecipitated with anti-HA antibody and visualized by immunoblot. (<b>H</b>) US2 induces integrin α4 ubiquitination. THP-1 cells expressing integrin α4-HA in combination with US2 or empty vector were pulse-chase labeled for the indicated times. Integrin α4 was immune precipitated using the HA-epitope tag, eluted from beads, fully denatured to dissociated non-covalently linked proteins and re-precipitated using α-HA or α-ubiquitin antibodies.</p