MEF and MNC were infected with Δm157-MCMV:luc (MOI as indicated) or were mock treated.

<p>At 4, 24 and 48 h post infection, protein lysates were generated. Viral gene expression was analysed by immunoblot analysis using MCMV-specific antibodies.</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

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

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

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

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

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

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

The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions

<div><p>Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus.</p></div