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

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

Generation of MHV-68 mutants.

<p>A) Schematic presentation of viral mutants. B) Scheme of the expected fragments after digestion of viral DNA with the restriction enzyme <i>EcoR</i>I. Digestion of DNA from both parental virus and the 40 bp revertant with <i>EcoR</i>I results in a 5.2 kb wildtype fragment. Deletion of the 40 bp repeat results in the loss of the 5.2 kb fragment and in the generation of a new 2.8 kb fragment. Partial loss of repeat units results in a shift of the 5.2 kb fragment to 4.6 kb and 4.2 kb fragments, respectively. “P” indicates the probe used for Southern blot analysis, corresponding to nucleotides 25889-26711. C) Structural analysis of reconstituted virus genomes by ethidium bromide-stained agarose gel analysis of viral DNA digested with <i>EcoR</i>I. Lane 1: Parental virus; Lane 2: 40 bp revertant; Lane 3: Delta 40 bp mutant; Lane 4: 40 bp moderate mutant; Lane 5: 40 bp low mutant; Lane 6: M6STOP mutant. D) Southern blot analysis of the gel shown in panel C using probe “P” indicated in panel B. The expected fragments are indicated by dots (panel C) or by arrows (panel D). Marker (M) sizes (in kilobase pairs) are indicated on the left.</p

The 40 bp internal repeat is important for virus-induced splenomegaly and for <i>ex vivo</i> reactivation of latently infected splenocytes.

<p>C57BL/6 mice were i.n. infected with 5×10⁴ PFU. A) Extent of splenomegaly. At the indicated time points after infection, spleens were harvested and the spleen weight was determined. Shown are means±SD of the number of mice indicated in brackets. The asterisks indicate a statistically significant difference (p = 0.043 at day 13 and p = 1.02×10⁻⁸ at day 17; Student's t-test). The extent of <i>ex vivo</i> reactivation was determined 10 (B), 13 (C), 17 (D) and 23 (E) days after infection. Data shown are from a representative experiment with splenocytes pooled from 3 mice per group. F) Summary of independent <i>ex vivo</i> reactivation experiments performed with splenocytes at day 17 after infection. In each individual experiment, splenocytes from 3 mice per group were pooled. Shown are the reciprocal frequencies of reactivation (mean±SD; n = 4 for parental virus and Delta 40 bp mutant, n = 2 for revertant), calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000733#s2" target="_blank">materials and methods</a>. The asterisk indicates a statistically significant difference (p = 0.040; Student's t-test).</p

Viral genomic load in the spleen.

<p>Total DNA was extracted from spleens at days 17 and 42 after infection. Real time PCR was performed with 100 ng of total splenocyte DNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000733#s2" target="_blank">materials and methods</a>. Data shown are mean values from 3 to 4 individual mice, each tested in duplicate, compiled from two independent experiments. The asterisk indicates a statistically significant difference (p = 0.024; Student's t-test).</p

The 40 bp internal repeat is dispensable for lytic replication in the lung.

<p>C57BL/6 mice were i.n. infected with 5×10⁴ PFU. At the indicated time points after infection, lungs were harvested and the virus titers of lung homogenates were determined by plaque assay on BHK-21 cells. Each symbol represents a single mouse. The dashed line indicates the limit of detection which is 50 PFU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000733#pone.0000733-Adler2" target="_blank">[31]</a>.</p