HSV-1 induces a bona fide transactivaion of the pTA-Control promoter.

<p>(<b>A</b>) Vero cells were transfected with the pTA-Control plasmid. The cells were split and infected with HSV-1 F strain. 24 h p.i. the expressed luciferase activity was measured. The cells were lysed and DNA was extracted in parallel, separated on an agarose gel and used for a Southern blot. The membrane was probed with DIG labelled (DIG high prime, Roche) pTA-Control plasmid. (<b>B</b>) CV-1 cells were transfected with 700 ng of the pTA-Control vector. Cells were infected with 10 PFU/cell HSV-1 F strain. Cells were incubated for 4 h with cycloheximide (CHX), which was replaced by 5 µg/ml actinomycin D (ActD) 4 h p. i. for selective immediate early protein expression conditions. Absence of ActD following washing (fifth bar f. l.) documents reversibility of CHX and continuous CHX and ActD treatment served as control for drug efficacy. Cells were lysed and luciferase activity was determined.</p

The episomal vector pB45Neo-promLuc confers herpesvirus responsiveness.

<p>(<b>A</b>) HeLa cells were transiently transfected with the pB45Neo-promLUC construct by nucleofection. Cells were infected with HSV-1 F strain (0.5 or 3 PFU/cell) or left uninfected. Cells were lysed and luciferase activity was determined. (<b>B</b>) G418-selected stable M2-10B4 cell clones (#1, #3 and #4) were infected with 5 PFU/cell HSV-1 F strain for 16 hours. Cells were lysed and luciferase activity was determined.</p

Herpesviruses transactivate the pTA-luc plasmid series.

<p>(<b>A</b>) Human MRC-5 cells, transiently transfected with the pTA-Control plasmid (left panel) or pTA-GAS (right panel), were split into aliquots and seeded. Cells were infected with 1 PFU/cell HCMV-AD169, HCMV-Towne, HCMV-TB40/E (black bars) or left uninfected (white bars). After 24 h the cells were treated for additional 5 h with 500 U/ml human IFN-γ. Cells were lysed and luciferase activity was measured. The experiment was performed in triplicate and the arithmetic mean with the standard deviation is shown. The significance was tested with the Student's t-test: * p<0.05 and ** p<0.01. (<b>B</b>) ARPE-19, CV-1, Vero and Jurkat cells were transfected with 2.5 µg pTA-Control plasmid using the Lonza nucleotransfection system. Cells were split so that parallel measurements and infections originated from the same transfection. 20 h post transfection cells were left uninfected (mock; open bars) infected with 3 PFU/cell HCMV-AD169, HSV-1 F strain (black bars) or UV-inactivated virus (hatched bar) for additional 20 h. Cells were lysed and luciferase activity was measured. (<b>C</b>) MRC-5 cells were infected with 3 PFU/cell HCMV-AD169 or UV-inactivated HCMV-AD169. After the indicated time cells were lysed and total RNA or protein was prepared. Protein lysates were subjected to native sodium-deoxycholate-PAGE for the analysis of IRF-3 dimerization or to SDS-PAGE for the analysis of pp72-IE1 and β-actin protein amount. Proteins were transferred to filters and probed with the indicated antibodies. IFN-β and GAPDH mRNA was assessed by semi-quantitative RT-PCR from total RNA as. For GAPDH two log₁₀ dilutions were used as template to confirm measurement in the linear amplification range.</p

Time course of luciferase induction upon infection with HSV-1 and HCMV.

<p>MRC-5 cells were transfected with the pTA-Control plasmid by nucleofection and subsequently infected with HCMV-HB5 or HSV-1 F strain (4 PFU/cell). PAA was added at the time point of infection. Cells were lysed at indicated time points post infection and luciferase activity was measured.</p

Virus-induced transactivation can be neutralized using IVIG.

<p>(<b>A</b>) MRC-5 cells were transfected with 2 µg pTA-Control plasmid using the Lonza transfection protocol and reagents. Cells were infected at different PFU/cell (10; 5; 2; 0.5 or 0.1 PFU/cell) of infectious HCMV-HB5 (black bars), UV-inactivated HCMV-HB5 (hatched bar) or left uninfected (white bar). Virus was incubated with indicated dilutions of the IVIG preparation Cytotect® for 1 h at 37°C before infection. As control the virus was incubated with the pooled sera of two seronegative donors (grey bar). (<b>B</b>) As in (<b>A</b>), but CV-1 cells were used and infected with HSV-1 strain F (2 PFU/cell). (<b>C</b>) 5 PFU/cell HSV-1 F strain was incubated for 90 min at 37°C with 1/10, 1/20 or 1/100 vol/vol dilutions of 14 human sera. #N1–#N7 HSV-1 seronegative donors and #P1–#P7 HSV-1 seropositive donors. pB45Neo-promLUC transfected HeLa cells were infected with virus-serum suspensions. 20 h post infection cells were lysed and luciferase activity was determined.</p

pTA-Control is transactivated upon herpesvirus infection in a virus dose-dependent manner.

<p>(<b>A</b>) WI-38 cells were transiently transfected (nucleofection) with the pTA-Control plasmid. Cells were infected for 14 h (left panel) or 24 h (right panel) with indicated PFU/cell HCMV-TB40/E. Cells were lysed and luciferase activity was measured. The arithmetic mean of triplicates ± standard deviation (SD) is shown. Statistical significance was tested by unpaired two-sided t-test with unequal variance compared to the mock (black asterisks) and the corresponding lower infectious dose (depicted in grey). * p<0.05; **p<0.01 and *** p<0.001. Please note the different scale between left (*10⁴) and right (*10⁵) panel. (<b>B</b>) As in (<b>A</b>) but HeLa cells were nucleofected with pB45Neo-promLUC and infected with the indicated infectious dose of HSV-1 F strain (black bars) or a low-passage clinical HSV-1 isolate (grey bars). Arithmetic mean of triplicates ± SD is shown. p-values as in (A).</p

Specificity of responsiveness of the pTA-Control and the pB45Neo-promLUC construct.

<p>(<b>A</b>) HeLa cells were transiently transfected with 2 µg of the ‘6× κB-Luc’ NF-κB-reporter construct, the pTA-Control or the pB45Neo-promLUC. 16 h later cells were treated with 20 ng/ml tumour necrosis factor (TNF)-α, 5 ng/ml interleukin (IL)-1β, 0.1 µg/ml phorbol-12-myristate-13-acetate (PMA) or 1 µg/ml ionomycin (Iono) or both (PMA/Iono) for 4.5 h. A 16 h infection with 10 or 1 PFU/cell HSV-1 F strain (HSV 1 and HSV 10, respectively) served as control for inducibility by herpes viruses. (<b>B</b>) HeLa cells were transiently transfected with the pTA-Control or the pB45Neo-promLUC construct and infected with 0.1, 1 or 10 TCID50 vesicular stomatitis virus (VSV) and 0.1, 1 or 10 PFU/cell HSV-1 F strain, respectively. 15 h later cells were lysed and luciferase activity was measured. (<b>C</b>) 10⁶ RPMI8866 cells were transfected with 2 µg of the pTA-Control construct and infected over night with the indicated EBV genome copies/cell. Cells were lysed and luciferase activity was determined.</p

Viral transactivation can be neutralized by monoclonal antibodies in MRC-5 and Jurkat cells.

<p>(<b>A</b>) MRC-5 cells were transiently transfected (Lipofectamine 2000 CD) with the pTA-Control plasmid. Cells were infected with HCMV-HB5 (left panel) or HSV-1 strain F (right panel). Replication competent virus (black bars) was compared with UV-inactivated virus (hatched bars). HCMV was incubated for 1 h at 37°C before infection with 25 µg/ml of the HCMV gB-specific monoclonal antibody ITC88 and HSV-1 with the HSV-1 gD-specific monoclonal antibody HD1 before infection. Cells were lysed and luciferase activity was determined. (<b>B</b>) Jurkat cells were transfected with pTA-Control plasmid using the Lonza transfection reagents and protocol. Cells were infected with UV-inactivated HSV-1 strain F (hatched bar), replication competent HSV-1 (black bars) or left uninfected (white bar). Virus was incubated before infection for 1 h at 37°C with indicated dilutions of HSV gD monoclonal antibody HD1. ∼20 h p. i. cells were lysed and the expressed luciferase activity was measured.</p

Ectopic expression of HSV-1 gE, HCMV gp68 and HCMV gp34 inhibit IgG1 (trastuzumab) mediated activation of FcγRs.

<p>(<b>A</b>) SKOV-3 cells were infected for 24 h with 2 PFU/cell VACV wt and rVACV expressing gE or (<b>B</b>) rVACV expressing gp68 or gp34 before opsonized with trastuzumab at different concentrations for 30 min. After removing of unbound antibodies by repeated washing with D-MEM 10% (vol/vol) FCS, 1×10⁵ BW:FcγR-ζ transfectants per well were added and co-cultivated overnight. BW:FcγR-ζ activation was determined by measuring mIL-2 by ELISA. Three independent replicates were measured, means with standard deviations (error bars) are shown for 3 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

Soluble ectodomains of HCMV vFcγR interfere with antibody dependent NK cell degranulation.

<p>(<b>A</b>) Cytotect was coated to a plate in binding buffer (0.1 M Na₂HPO₄ pH 9.0) at a concentration of 0.5 mg/ml and incubated for 2.5 hours at 37°C. After blocking for 30 minutes and washing unbound antibodies, soluble proteins, rIL-2 pre-activated primary NK cells and α-CD107a-PECy5 antibody were added and incubated for 4 hours at 37°C. Duplicates were measured for CD107a surface expression after dead cell exclusion with DAPI staining in a FACS Canto II. Means are shown with standard deviations (error bars). Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001. (<b>B</b>) To compare the amounts of soluble proteins used in (A), SDS-PAGE and anti-V5 immunobotting was performed.</p