mLANA mediates kTR episome persistence.

<p>Gardella gel after transfection of A20 or A20/mLANA cells with pRepCK vector or k8TR DNA. Lanes contain 2-3x10⁶ cells. Gel was performed at 24 days of puromycin selection. Blot was probed with ³²P-pk8TR DNA. O, gel origin; E, S11 episomes; L, S11 linear genomes due to lytic replication; ccc plasmid DNA is indicated.</p

Episome maintenance of mTR or kTR DNA in mLANA or kLANA expressing cells.

<p>Episome maintenance of mTR or kTR DNA in mLANA or kLANA expressing cells.</p

v-kLANA latent infection.

<p>Viral latency in spleens of C57 BL/6 mice 14 days after i.n. infection with 10⁴ PFU of the indicated viruses. (A) Latent titers determined by co-culture reactivation assay (closed circles) and titers of pre-formed infectious virus by plaque assay (open circles). Circles are titers of individual mice. Bars indicate mean and dashed line shows the limit of assay detection. v-kLANA titers were significantly lower than v-WT (Mann-Whitney test). *p<0.05. (B) Quantification of viral DNA-positive cells in total splenocytes and in sorted GC B cells (CD19⁺CD95⁺GL7⁺). Data are from pools of five spleens per group. Bars are frequency of viral DNA-positive cells. Error bars indicate 95% confidence intervals. (C-E) Flow cytometry analyses. Representative FACS plots from individual mice are shown in left panels. Quantification graphs in which each point represents an individual mouse are shown at the right. Bars are mean values. Data were combined from 2 independent experiments with 5 mice in each group. (C) Total number of GC B cells (CD19⁺CD95⁺GL7⁺). NS, not significant; *p<0.05 using the Mann-Whitney test. (D) Percentage of GC B cells that were YFP positive. (E) Percentage of YFP positive cells that were GC B cells. ***p<0.001 in (D) and (E) using the Mann-Whitney test.</p

mLANA and kLANA expression in vivo.

<p>Spleen sections of mice infected with 10⁴ PFU of v-WT or v-kLANA for 14 days. (A) In situ hybridization (brown) with probes for viral miRNAs 1–6. Sections were counter stained with Mayer´s Haemalum. (B, C) Detection of kLANA (B) and mLANA (C) by immunohistochemistry in sections adjacent to those shown in panel A. Arrows in panel B indicate the same kLANA positive cell. Arrow in panel C indicates a mLANA positive cell. Sections were counterstained with haematoxylin. No kLANA signal was detected in sections stained only with secondary antibody. (D, E) mLANA and kLANA nuclear dots detected by indirect immunofluorescence. Images are maximum intensity projections of Z-stacks acquired over the thickness of the spleen sections. No dots were observed in unstained sections or with secondary antibody alone. Magnification 630x. (F) Quantification of mLANA (n = 69 nuclei from 3 mice) or kLANA (n = 67 nuclei from 3 mice) dots per 100 μm³ nuclear volume. Bars indicate means. The number of dots per volume was not significantly different between v-WT and v-kLANA mice (Mann-Whitney test, p>0.05). (G) Viral genomes in FACS sorted YFP⁺ and YFP^- GC B cells from spleens of v-WT.yfp (n = 7) and v-kLANA.yfp (n = 6) infected mice. Circles represent individual mice. Bars indicate means. There was no significant difference between the two infection groups (Mann-Whitney test, p>0.05).</p

kLANA mediates mTR episome persistence.

<p>(A) Schematic of kLANA and mLANA. Homologous regions are indicated in grey shading. White and black regions share no homology. Amino acid residue numbers are indicated. P, proline-rich. Gardella gels after transfection of m4TR (B) or m8TR (C) DNA. Blots in B and C were probed with ³²P-pRepCK DNA. (D) Gardella gel after 87 days of indicated cell lines from panels B and C. Blot was probed with ³²P-m8TR DNA. Days of G418 selection are below each panel. O, gel origin; ccc plasmid DNA is indicated. Lanes contain 1.5-2x10⁶ cells. Vertical lines at right (panels B, C, E) indicate positions of episomal bands. Asterisks indicate faint episomal bands. (E) Immune fluorescence for kLANA. m8TR cells are from cell line d (panels C, D). Brightness and contrast were uniformly adjusted in panels from the same field and red signal was uniformly enhanced for k8TR panels using Adobe Photoshop. Magnification, 630x.</p

Generation and lytic growth of MHV68 chimeric viruses.

<p>(A) Schematic diagram. The kLANA cassette was inserted between the M11 stop codon and the mORF72 exon in place of MHV68 103,935–104,709, which includes most of the mLANA ORF. p1, p2, p3, are mLANA promoters[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006555#ppat.1006555.ref038" target="_blank">38</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006555#ppat.1006555.ref039" target="_blank">39</a>]. The mORF72 noncoding exon (black) is located within the mLANA coding region. The E2 splice acceptor site (nt 104,871) and the mORF72 exon splice donor site (nt 104,715) were left intact to ensure expression of kLANA and mORF72. The mLANA start codon and three downstream ATGs were mutated to ATT to prevent initiation of translation (indicated by black dots). The BamHI-G fragment (genomic nt 101,653–106,902) is indicated. mLANA ORF, nt 104,868–103,927. (B) Confocal immunofluorescence detection of mLANA (top panels) or kLANA (lower panels) from yfp viruses. Magnification 630x. (C) Immunoblot of viral proteins. (D) Growth curves of virus in BHK-21 cells after infection with 0.01 PFU/cell. There was no significant difference between infection groups (p>0.05 using one-way non-parametric ANOVA Kruskal-Wallis). (E) Lung virus titers 7 days after infection with 10⁴ PFU of the indicated viruses. Circles represent titers of individual mice (n = 19). Bars indicate the mean. v-Δ1007-21.yfp had significantly lower titers than v-WT.yfp (**p<0.01, using one-way non-parametric ANOVA Kruskal-Wallis followed by Dunn´s multiple comparison test). There were no other statistically significant differences between groups.</p

Interaction of mLANA_124–272 and mLANA_124–272 H186D/K187E with TR-DNA.

*<p>Full-length mLANA protein used in this experiment.</p

mLANA DNA binding is essential for virus persistence and the dorsal positive patch exerts a role in the expansion of GC B cells.

<p>(<b>A</b>) Amino acid substitutions in recombinant viruses (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003673#ppat.1003673.s005" target="_blank">Figure S5</a>). (<b>B</b>) Infection of BHK-21 cells at 0.01 p.f.u. per cell. Virus titres were determined by plaque assay. (<b>C</b>) Lungs from infected mice were removed and infectious viruses were titrated by plaque assay. (<b>D and E</b>) Quantification of latent infection in spleen by explant co-culture plaque assay (closed circles). Titres of infectious virus were determined in freeze/thawed splenocyte suspensions (open circles). Each circle represents the titre of an individual mouse. The dashed line represents the limit of detection of the assay. Mutant viruses are shown in panel D and revertant viruses in panel E. (<b>F and G</b>) Reciprocal frequencies of viral DNA-positive cells in total splenocytes (<b>F</b>) or GC B cells (CD19⁺CD95^hiGL7^hi) (<b>G</b>) were determined by limiting dilution and real-time PCR. Data were obtained from pools of five spleens per group. Bars represent the frequency of viral DNA-positive cells with 95% confidence intervals. (<b>H</b>) Identification of latently infected cells in spleens by <i>in situ</i> hybridization. Representative splenic sections from each group of viruses are shown. All images are magnified ×200. Dark staining indicates cells positive for virally encoded miRNAs (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003673#ppat-1003673-t004" target="_blank">Table 4</a>).</p

Data collection and refinement statistics.

<p><i>R</i>_work and <i>R</i>_free are defined by <i>R</i> = ∑<i>hkl</i>∥<i>F_obs</i>|−|<i>F</i>_calc∥/∑<i>hkl</i>|<i>F</i>_obs|, where <i>hkl</i> are the indices of the reflections (used in refinement for <i>R</i>_work; 5%, not used in refinement, for <i>R</i>_free), and <i>F</i>_obs and <i>F</i>_calc are the structure factors deduced from measured intensities and calculated from the model, respectively. Values in parentheses are for the highest resolution shell.</p

Kinetic values for mLANA_124–314 with TR-DNA by ITC.

<p>Standard deviations were calculated from three independent measurements. Gibbs free energy and entropy were calculated using these equations ΔG = RT lnK_D and ΔG = ΔH−TΔS.</p