Chemokine Binding Protein M3 of Murine Gammaherpesvirus 68 Modulates the Host Response to Infection in a Natural Host

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology

Influence of M3 on the changes in chemokine levels in the lungs of infected wood mice.

<p>Protein from lung lysates of wood mice infected with M3.stop or M3.MR MHV-68 were analysed for the concentration of specific chemokines by ELISA. Data were normalized relative to total protein concentration as determined by a modified Bradford assay. Error bars depict the standard error of the means for 4 individual wood mice. Statistical analysis was performed using Student's <i>t</i>-test and where significance was found this is indicated above the bars.</p

Influence of M3 on T and B cell numbers in the lungs of acutely infected wood mice.

<p>All data were from mice infected with either M3.MR or M3.stop virus at either 7 or 14 days p.i. as indicated. Quantification of B and T cell infiltrates was performed by counting the number of cells in corresponding sequential sections from the same histopathological sections as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001321#ppat-1001321-g003" target="_blank">Fig. 3</a>. The number of cells per unit area (138138 µm²) was then calculated for the interstitial infiltrate (Panels A and B) and the proportion of B and T cells for each area of perivascular/peribronchiolar (PV/PB) infiltration (Panels C, D) and iBALT (Panel E). Data are shown as the mean values ± SEM and compared between groups using a two sample <i>t</i>-test.</p

Comparative analysis of <i>M3</i> RNA expression in lungs of infected wood mice.

<p>Quantification by qRT-PCR of RNA expressed from the MHV-68 genome within lungs; <i>ORF50</i> RNA levels were assessed as a general reference for lytic-cycle gene expression. The copy numbers of individual viral-gene RNAs (as cDNAs) were normalized to those for cellular <i>RPL8</i>. Error bars represent the standard error of the mean from three wood mice per time point. (A) analysis of expression from the <i>M1-M4</i> locus of infected wood mice lungs. (B) analysis of M3 expression from the lungs of BALB/c and wood mice. Note that, although <i>M3</i> expression is similar for both species at 7 days p.i., after 14 days p.i., <i>M3</i> expression in BALB/c mouse lungs is drastically reduced compared to wood mice.</p

Contribution of M3 to MHV-68 infection in wood mice.

<p>(A) Loss of M3 is associated with reduction of viral DNA in lung at 7, 14 and 40 days p.i. Quantitative PCR analysis of viral genome copies per 200 ng lung DNA. (B) Reduction in latently infected cells in the spleen at 14 days p.i. as a consequence of M3 loss, as determined by infective center assay. (C) Reduction in viral DNA in spleen at 14 and 40 days p.i. parallels the loss of latently infected splenocytes in (B). Quantitative PCR analysis of viral genome copies per 200 ng spleen DNA. Data in (A) and (C) represent the mean value determined from three individual wood mice per infection, normalized to copies of cellular <i>rpl8</i> per DNA sample; error bars depict the standard error of the means. Statistical analysis was performed using Student's <i>t</i>-test and where significance was found this is indicated above the bars.</p

Influence of M3 in spleen of acutely infected wood mice.

<p>All data were from mice infected with either M3.MR or M3.stop virus at 14 days p.i. (A) In M3.MR infected mice, the white pulp is composed of secondary follicles with distinct germinal centers, exhibiting obvious light and dark zones. HE stain. (B) In M3.stop-infected mice, follicles are larger and exhibit large, poorly delineated germinal centers. HE stain. (C) Localization, by <i>in situ</i> hybridization to <i>vtRNAs</i>, of latently infected splenocytes within spleen of M3.MR-infected mice. Latently infected cells are primarily contained within the light zone of germinal centers. (D) <i>vtRNA</i> detection within latently infected splenocytes in M3.stop-infected mice. Note that positive cells are found scattered throughout the follicle, as well as in the red pulp. Labels: F, follicle; GC, germinal center; RP, red pulp.</p

Array analysis of changes in pulmonary chemokine and cytokine levels within infected wood mice as a consequence of M3 loss.

<p>Equal amounts of protein from lung lysates of wood mice infected with M3.stop or M3.MR MHV-68 were incubated with RayBiotech 3.1 membrane arrays capable of detecting 61 different chemokines and cytokines. Shown are the relative abundance of cytokines and chemokines (minus background) whose expression consistently showed more than a two-fold difference in M3.stop- versus M3.MR-infected mouse lung lysate (two independent experiments). Asterisks denote chemokines that have been tested <i>in vitro</i> and found to be bound by M3; # denotes chemokines tested and not bound by M3, according to van Berkel <i>et al... </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001321#ppat.1001321-vanBerkel2" target="_blank">[16]</a> and Parry <i>et al</i>. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001321#ppat.1001321-Parry1" target="_blank">[15]</a>; + denotes that MIP-3/CCL19-dependent chemotaxis is inhibited by M3 according to Jensen <i>et al... </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001321#ppat.1001321-Jensen1" target="_blank">[46]</a>; #* denotes that in the case of BLC/CXCL13, while Parry <i>et al</i>. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001321#ppat.1001321-Parry1" target="_blank">[15]</a> and Martin <i>at al</i>. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001321#ppat.1001321-Martin1" target="_blank">[62]</a> found that M3 bound weakly and inhibited factor-dependent chemotaxis, van Berkel <i>et al... </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001321#ppat.1001321-vanBerkel2" target="_blank">[16]</a> did not observe any binding to M3.</p

Oligodeoxynucleotide primers used.

<p>Oligodeoxynucleotide primers used.</p

Influence of M3 in lung of acutely infected wood mice.

<p>All data were from lungs of mice infected with either M3.MR (left panels) or M3.stop virus (right panels) at 14 days p.i. (A) M3.MR (i.e., pseudo wild-type MHV-68) infected wood mouse lung tissue (HE stain) showing large perivascular lymphocyte infiltrations; arrows indicate iBALT. (B) M3.stop infected wood mouse lung showing much smaller perivascular lymphocyte infiltration (arrow). Panels (C) and (D): Immunohistological staining showing B-cell (CD45R⁺) dominance in the peribronchial infiltrations (iBALT) of mice infected with M3.MR (C; arrows), and the reduced proportion of B cells in the equivalent infiltrates in mice infected with M3.stop virus (D; arrow). Panels (E) and (F): Immunohistological staining for T cells (CD3⁺) indicating that they are a minority cell type in iBALT within M3.MR-infected mice (E; arrows), whereas T cells are more prevalent in the infiltrates within M3.stop-infected mice (F; arrow); T cells were also seen rolling along endothelial walls (F; inset and arrows). Panels (G) and (H): Detection by <i>in situ</i> hybridization of <i>vtRNA</i> expression (indicative of latent infection). In mice infected with M3.MR, positive cells are numerous in the perivascular infiltrates (G; arrows) and are seen attached to the endothelial wall (G; inset and arrows). They are present but less numerous in mice infected with M3.stop (H; arrow). All images are representative of numerous tissue sections analyzed from 3 wood mice per infection. Labels: A, artery; V, vein; B, bronchiole.</p

Localization of <i>M3</i> expression in lung, spleen, and lymph node.

<p>Detection of <i>M3</i> RNA in lung and spleen by <i>in situ</i> hybridization in MHV-68 infected wood mice. A, B: Lung, day 7 p.i.: (A) <i>M3</i> transcripts detected within lymphocytes in perivascular infiltrates (white arrows) and lymphocytes attached to the endothelial wall (black arrow). A: artery; (B) No signal detected with sense-strand probe (negative control). C, D: Lung, day 12 p.i.: (C) Perivascular and peribronchial lymphocyte infiltrations containing numerous <i>M3</i>-positive lymphocytes. A, artery; B, bronchioles; (D) Peribronchial, focal follicle-like lymphocyte accumulation with numerous <i>M3</i>-positive lymphocytes. B, bronchiolus. (E) Bronchial lymph node, day 7 p.i.; lymphocytes showing <i>M3</i> transcripts are present in lymphatic follicles (within germinal center cells). F, follicle. (F) Spleen, day 14 p.i.; follicle with numerous <i>M3</i> RNA-positive lymphocytes in the germinal center. Results are representative of numerous tissue sections analyzed from three infected wood mice.</p