Analysis of the influence of Rbbp4 on lytic virus replication.

<p>To analyze lytic growth of recombinant viruses expressing Rbbp4, TCMK-1 cells were plated and after 24 hours infected with the indicated viruses at an MOI of 0.01 for 1 hour. After removing the inoculum, cells were incubated with fresh medium at 37°C and 5% CO₂ until the supernatants together with the cells were harvested at the indicated time points after infection. Virus titers were determined by plaque assay. The means + SD of three independent experiments are shown (A and E). BHK-21 cells were plated and after 24 hours transfected with 2 μg of the indicated BAC plasmid DNA. Plaque development and cytopathic effect (virus reconstitution) were monitored by light microscopy, and photomicrographs were taken at the indicated time points. Scale bar, 500 μm (B). Cell free supernatants from (B) were harvested and virus titers were determined by plaque assay. Data shown are means + SD from duplicate determinations (C). BAC plasmid DNA replication was measured by real-time PCR analysis after co-transfection of a BAC plasmid lacking the right oriLyt with an Rbbp4-expression plasmid or a control plasmid. Data shown are means + SD from three independent experiments (D).</p

Lytic replication in the lungs after i.n. inoculation.

<p>C57BL/6 mice were inoculated i.n. with 1x 10³ PFU of the indicated viruses. Lungs of mice were harvested at day 3 after infection (parental virus or mutant viruses) and at day 6 after infection (parental or mutant viruses and ectopic revertants). Virus titers were determined from organ homogenates by plaque assay. Each symbol represents an individual mouse, and the bars represent the median value. (A) The data are compiled from two (day 3) and five (day 6 mutant viruses) or six (day 6 parental virus) independent experiments. (B and C) The data are compiled from two independent experiments in each case. The asterisks indicate a statistically significant difference between the groups (* P < 0.05; ** P < 0.01; *** P < 0.001).</p

Proteins associated with the right oriLyt as identified by DNA-affinity purification and mass spectrometry analysis^a.

<p>Proteins associated with the right oriLyt as identified by DNA-affinity purification and mass spectrometry analysis<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005510#t001fn001" target="_blank">^a</a>.</p

Confirmation of up- or downregulation of Hexim1.

<p>Upregulation of Hexim1 was accomplished by stimulation of NIH 3T3 cells with HMBA, and the expression of Hexim1 was analyzed by quantitative PCR on mRNA level (A) and by Western Blot on protein level (B). In addition to the predicted 54 kDa band, a second band at approximately 40 kDa was detected by Western Blot with lysates from NIH 3T3 cells, probably representing a modified form of the protein. To downregulate Hexim1, stable cell lines of TCMK-1 cells expressing shRNA specific for Hexim1 were generated. Downregulation of Hexim1 in these cell lines was confirmed by quantitative PCR (C) and by Western Blot (D). The shRNAs 1 and 2 proved to be most effective regarding downregulation of Hexim1.</p

Latent infection in PECs after i.p. inoculation.

<p>C57BL/6 mice were inoculated i.p. with 1x 10⁴ PFU of the indicated viruses. PECs were harvested at day 17 after infection to analyze latent infection in the ex vivo reactivation assay (A and B) or for DNA isolation and real-time PCR analysis of the viral genomic load (C and D). Data for parental virus and virus mutants are obtained from two independent experiments, each with five mice per group. Data for the ectopic revertants (ER Δright oriLyt and ER Δleft oriLyt) are obtained from a single experiment with five mice per group. For the ex vivo reactivation assay, cells from five mice per group were pooled in each experiment and data shown are the means ± SEM (parental virus or virus mutants) or values from a single experiment (ectopic revertants). The dashed line indicates the point of 63.2% Poisson distribution, determined by nonlinear regression, which was used to calculate the frequency of cells reactivating lytic replication. In panels C and D, each symbol represents an individual mouse, and the bars represent the median value. Asterisks indicate a statistically significant difference between the groups (* P < 0.05).</p

Virus replication in vitro after upregulation or downregulation of Hexim1.

<p>To upregulate Hexim1, NIH 3T3 cells were plated and after 4 hours stimulated with 5 mM or 10 mM HMBA or left untreated. 24 hours later, cells were infected with the indicated viruses at an MOI of 0.01 for 1 hour. After removing the inoculum, cells were incubated with fresh medium without HMBA (A), with 5 mM HMBA (B) or with 10 mM HMBA (C) at 37°C and 5% CO₂ until the supernatants together with the cells were harvested at different time points after infection. Virus titers were determined by plaque assay. For the “no treatment” control, the means ± SEM of duplicates from three independent experiments are shown. Data shown for the HMBA treated cells are from three independent experiments showing each single experiment. To downregulate Hexim1, TCMK-1 cells were stable transfected with two different shRNAs specific for Hexim1 or with a scrambled control shRNA, respectively (D). Cells were plated and after 24 hours infected with the indicated viruses at an MOI of 0.01 for 1 hour. After removing the inoculum, cells were incubated with fresh medium at 37°C and 5% CO₂ until the supernatants together with the cells were harvested at different time points after infection. Virus titers were determined by plaque assay. For the “scrambled shRNA” control, the means ± SEM of duplicates from two independent experiments are shown. For the shRNAs specific for Hexim1, the means ± SD of two independent experiments each are shown.</p

Confirmation of results from DNA-affinity purification and MassSpec.

<p>Formaldehyde cross-linking ChIP assays were performed to ensure that Hexim1 and Rbbp4 are associated with DNA of the right oriLyt of MHV-68. The immunoprecipitates isolated from TCMK-1 cells by a specific antibody against Hexim1 (A) or from NIH 3T3 cells by a specific antibody against Rbbp4 (B) were analyzed by quantitative PCR with primer pairs designed to amplify a part of the DNA sequence of the right oriLyt and the left oriLyt, respectively, or by a primer pair amplifying a non-relevant sequence located in the viral ORF23.</p

Identification of ORF4 gene products.

<p>A. NMuMG epithelial cells or BHK-21 fibroblasts were infected with wild-type (WT) or ORF4-deficient (ORF4⁻) MHV-68 (2 PFU/cell). 48 h later, cells were removed by low speed centrifugation (400×g, 10 min) and virions then removed by high speed centrifugation (20,000×g, 3 h). Supernatants were immunoblotted with mAbs 16D2 or 9C7. B. The wild-type virions from A were similarly analyzed by SDS-PAGE and immunoblotting. Neat and 1/3 diluted lysates are shown. C. Virus from BHK-21 (B) or NMuMG cells (N) was denatured and then left undigested (nil) or treated with PNGase F (N-gase) or sialidase plus O-glycanase (O-gase). All samples were resolved by SDS-PAGE and immunoblotted with mAbs 9C7 or 16D2.</p

Virus replication in vitro.

<p>MOVAS (A), MHEC (B), SVEC 4–10 (C), TCMK-1 (D), MH-S (E) and CS16 stromal cells (F) were infected with the indicated viruses at an MOI of 0.01 (MOVAS, MHEC, SVEC 4–10, TCMK-1, CS16) or 1 (MH-S). Cells and cell culture supernatants were harvested at different time points p.i., and titers were determined by plaque assay on BHK-21 cells. Data shown are the means ± SD from three (SVEC 4–10 and MH-S), four (MOVAS and MHEC) or five (TCMK-1 and mMSC CS16) independent experiments.</p

Identification and characterization of mAbs specific for MHV-68 ORF4 gene products.

<p>A. MAbs from MHV-68-infected mice were used to stain either untransfected CHO-K1 cells (CHO) or CHO-K1 cells transfected with an ORF4 expression construct (CHO-ORF4). These cells were not cloned. Approximately 50% of them appeared to express ORF4. All ORF4-specific mAbs gave equivalent staining. T3B8 and T2B11 are shown as examples. nil = secondary antibody only. B. BHK-21 cells were either left uninfected (UI) or infected (18 h, 2 PFU/cell) with wild-type (WT) or ORF4-deficient (ORF4⁻) MHV-68. They were then trypsinized and analyzed for mAb binding by flow cytometry. Again, mAbs T2B11 and T3B8 are shown as representative examples. All ORF4-specific mAbs gave similar results except 9C7 (see D). C. Wild-type (WT) or ORF4-deficient (ORF4⁻) viruses were directly recovered from infected cell supernatants (virus) or also purified on density gradients (virions), denatured, resolved by SDS-PAGE and immunoblotted with mAbs 9C7 or 16D2. D. BHK-21 cells were left uninfected (UI) or infected with wild-type (WT) or ORF4-deficient (ORF4⁻) MHV-68 as in B, followed by flow cytometric analysis of mAb binding. E. BHK-21 cells were infected (18 h, 2 PFU/cell) with wild-type MHV-68, then labelled for 1 h with ³⁵S-cysteine/methionine. Viral proteins were immunoprecipitated from cell lysates with mAbs as indicated plus protein A-sepharose. A6, B12 and B9 are subclones of 9C7. T4C5 is a gH/gL-specific mAb. nil = protein A-sepharose only. The bands corresponding to gH and ORF4 product are indicated.</p