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

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

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

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

The absence of CD8⁺T cells partially reverses the phenotype of the Delta 40 bp mutant.

<p>A) Splenomegaly. C57BL/6 or CD8^−/− mice (5 mice per group) were i.n. infected with 5×10⁴ PFU. At day 17 after infection, spleens were harvested and the number of spleen cells was determined. Data shown are means±SD. The asterisk indicates a statistically significant difference (p = 0.01; Student's t-test). B) <i>Ex vivo</i> reactivation. C57BL/6 mice or CD8^−/− mice were i.n. infected with 5×10⁴ PFU. The extent of <i>ex vivo</i> reactivation was determined 17 days after infection. Splenocytes pooled from 5 mice per group were used.</p

The 40 bp internal repeat of MHV-68 is involved in latency amplification by regulating the expression of latency-associated genes.

<p>A) RT-PCR analysis of the expression of K3 after infection of fibroblasts (NIH3T3) and B cells (Ag8). As control, the expression of the murine ribosomal protein L8 gene, which was amplified in parallel, was determined. Lanes 1: Parental virus; Lanes 2: Delta 40 bp mutant; Lanes 3: Delta 100 bp mutant; M: marker. The sizes of the PCR products are indicated on the right. B) Quantitative RT-PCR analysis of the expression of K3 after infection of fibroblasts (NIH3T3) and B cells (Ag8). The data are presented as relative copy number of K3 to L8. C) Determination of the viral genomic load by PCR using primers specific for ORF 50 of MHV-68. As control, the murine ribosomal protein L8 gene was amplified in parallel. Lanes 1: Parental virus; Lanes 2: Delta 40 bp mutant; Lanes 3: Delta 100 bp mutant; M: marker. The sizes of the PCR products are indicated on the right. D) Quantitative PCR analysis of the genomic load after infection of fibroblasts (NIH3T3) and B cells (Ag8). The data are presented as relative copy number of ORF50 to L8. E) RT-PCR analysis of the expression of K3, ORF72 and ORF73 after infection of fibroblasts (NIH3T3) and B cells (Ag8). As control, the expression of the murine ribosomal protein L8 gene, which was amplified in parallel, was determined. Lanes 1: Parental virus; Lanes 2: Delta 40 bp mutant; Lanes 3: 40 bp revertant; M: marker. The sizes of the PCR products are indicated on the right. The data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000733#pone-0000733-g007" target="_blank">figure 7</a> are from representative experiments which were repeated 3 times with similar results.</p