Tyrosine 129 of the Murine Gammaherpesvirus M2 Protein Is Critical for M2 Function <i>In Vivo</i>

<div><p>A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes – predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe <i>in vitro</i> and <i>in vivo</i> studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells <i>in vitro</i>, only Y129 is critical for reactivation from latency and plasma cell differentiation <i>in vivo</i>.</p></div

Y120 and Y129 of M2 are required for expansion of primary murine B cells and for IL-10 production.

<p>(A and B) Primary murine B cells were transduced with retroviral vectors encoding Y120F or Y129F mutations of M2, wild type M2 or M2stop as a negative control. On days 2–5 post transduction, the cells were analyzed by (A) flow cytometry to monitor Thy1.1 expression, and (B) the supernatants from cells in (A) were analyzed for IL-10 secretion by ELISA. (C) 1–2×10⁶ cells each of the Y120F or Y129F inducible cell lines described in materials and methods were induced with doxycycline for 48 hours and supernatants were harvested for measurement of IL-10 secretion by ELISA. Data shown is a representative of one experiment with three replicates per condition. Each experiment was performed at least twice with at least three independent replicates per experiment.</p

Y129, but not Y120, of M2 is required for differentiation of infected B cells to plasma cells.

<p>(A–D) C57BL6 mice were infected with 1000PFU of the indicated virus either intranasally (A and C) or intraperitoneally (B and D) and splenocytes were harvested at indicated time points. Splenocytes were stained for flow cytometry as described in materials and methods. (A and B) Mice were harvested at the indicated time points after either IN (A) or IP (B) inoculation. YFP positive cells with a germinal center phenotype were analyzed as follows- doublet discriminated cells were gated on dump⁻ (a mix containing CD3, CD4 and CD8 antibodies to gate out T cells)/B220⁺/YFP+/CD95⁺GL7⁺. Data shown represents the frequency of Dump⁻/B220⁺/YFP⁺ cells that are CD95⁺GL7⁺, representing the infected cells with a germinal center phenotype. (C and D) Mice were harvested at the indicated time points after either IN (C) or IP (D) inoculation. YFP positive cells with a plasma cell phenotype were analyzed as follows- doublet discriminated cells were gated on dump⁻ (a mix containing CD3, CD4 and CD8 antibodies to gate out T cells)/YFP+/B220^l°CD138^hi. Data shown represents the frequency of Dump⁻/YFP⁺ cells that are B220^l°CD138^hi representing the infected cells with a plasma cell phenotype. Each data point refers to an individual mouse and the plots depict total mice from 3–4 experiments with 3–5 mice per group per experiment. <i>P</i> values were determined using a two-tailed paired Student’s <i>t</i> test (n.s., not significant).</p

M2 requires Y129, but not Y120, for efficient establishment of latency and reactivation from latency in splenic B cells.

<p>(A–D) C57BL6 mice were infected with 1000PFU of the indicated virus either intranasally (A–B) or intraperitoneally (C–D) and splenocytes were harvested at indicated time points. (A and C) The frequency of splenocytes harboring viral genomes were estimated by a limiting dilution, nested PCR assay as described in materials and methods. (B and D) The frequency of splenocytes reactivating virus in a limiting dilution <i>ex vivo</i> assay were analyzed on days 14–21 post plating, as described in materials and methods. For IN experiments (A and B), mice were harvested on 16–18 dpi and for the IP experiments (C and D) mice were harvested on 14–15 dpi. For the <i>ex-vivo</i> reactivation assay (B and D), intact cells were serially diluted and plated on a MEF monolayer alongside mechanically disrupted cells (as a measure of preformed infectious virus present), as described in materials and methods. The level of preformed infectious virus was below the limit of detection in the above experiments and is therefore not shown in the figures. Results shown in panels A and C are from 3 individual experiments with 3–5 mice per group. Results shown in panels B and D are from 4 individual experiments with 3–5 mice per group.</p

Infections with Y120F or Y129F mutant viruses do not have an effect on the total number of germinal center B cells or plasma cells.

<p>(A–F) C57BL6 mice were infected with 1000PFU of the indicated virus either intranasally (A, C and E) or intraperitoneally (B, D and F) and splenocytes were harvested at indicated time points. Splenocytes were stained for flow cytometry as described in materials and methods. (A and B) Mice were harvested at the indicated time points after either IN (A) or IP (B) inoculation. Splenocytes were analyzed for expression of YFP positive B cells after gating out doublet cells, followed by gating on dump⁻ (a mix containing CD3, CD4 and CD8 antibodies to gate out T cells) and B220⁺ cells. Data shown represents the frequency of Dump⁻/B220⁺/YFP⁺ cells. (C–F) Splenocytes were stained as above for determining the frequency of total number of cells with a germinal center phenotype, defined as dump^−/B220⁺/CD95⁺GL7⁺ cells in panel C and D or, total number of cells with plasma cell phenotype, defined as dump⁻/B220^l°CD138^hi cells in panel E and F. Each data point refers to an individual mouse and the plots depict total mice from 3–4 experiments with 3–5 mice per group per experiment. <i>P</i> values were determined using a two-tailed paired Student’s <i>t</i> test (n.s., not significant). Note for the data presented in panels C–F that there were no statically significant differences between experimental groups.</p

Y120F and Y129F do not induce phosphorylation of STAT3.

<p>(A–B) Primary B cells from wild type C57BL6 (B6) mice were transduced with retroviral vectors encoding either Y120F, Y129F, wild type M2 or M2stop. On days 2 and 3 post transduction, the cells were analyzed by phosphoflow as described in materials and methods. (A) B cells from B6 mice transduced as mentioned above were analyzed for pSTAT3 levels after gating on live cells by forward and side scatter characteristics. Representative histograms of pSTAT3 levels, one each on day 2 and day 3 are shown. (B) Mean Fluorescent Intensities (MFIs) of pSTAT3 fluorescence from three independent replicates are shown for each condition shown in A.</p

M2 induced IL-10 signals through positive feedback involving STAT3, but not STAT1 or STAT5.

<p>(A–F) Primary B cells from either wild type C57BL6 (B6) mice or IL10−/− mice (as indicated) were transduced with retroviral vectors encoding either M2 or M2stop. On days 2 and 3 post transduction, the cells were analyzed by phosphoflow as described in materials and methods. (A) B cells from B6 or IL10−/− mice transduced as mentioned above were analyzed for pSTAT3 levels after gating on live cells by forward and side scatter characteristics. Representative histograms of pSTAT3 levels, one each on day 2 and day 3 are shown. (B) Mean Fluorescent Intensities (MFIs) of pSTAT3 fluorescence from three independent replicates are shown for each condition shown in A. (C–D) B cells from B6 mice transduced as mentioned above were analyzed for pSTAT1 or pSTAT5 levels, respectively. Positive control for pSTAT1 induction is IFNγ treatment of cells at 100 ng/mL for 15 minutes and positive control for pSTAT5 induction is treatment of cells with GM-CSF at 100 ng/mL for 15 minutes. Representative histogram from day 2 post-transduction for each condition is shown. (E–F) Mean Fluorescent Intensities (MFIs) of pSTAT1 or pSTAT5 fluorescence from average of three independent replicates are shown for each condition shown in C and D. The experiments were done at least three times with three replicates in each experiment.</p

Gammaherpesvirus Co-infection with Malaria Suppresses Anti-parasitic Humoral Immunity

<div><p>Immunity to non-cerebral severe malaria is estimated to occur within 1-2 infections in areas of endemic transmission for <i>Plasmodium falciparum</i>. Yet, nearly 20% of infected children die annually as a result of severe malaria. Multiple risk factors are postulated to exacerbate malarial disease, one being co-infections with other pathogens. Children living in Sub-Saharan Africa are seropositive for Epstein Barr Virus (EBV) by the age of 6 months. This timing overlaps with the waning of protective maternal antibodies and susceptibility to primary <i>Plasmodium</i> infection. However, the impact of acute EBV infection on the generation of anti-malarial immunity is unknown. Using well established mouse models of infection, we show here that acute, but not latent murine gammaherpesvirus 68 (MHV68) infection suppresses the anti-malarial humoral response to a secondary malaria infection. Importantly, this resulted in the transformation of a non-lethal <i>P</i>. <i>yoelii</i> XNL infection into a lethal one; an outcome that is correlated with a defect in the maintenance of germinal center B cells and T follicular helper (Tfh) cells in the spleen. Furthermore, we have identified the MHV68 M2 protein as an important virus encoded protein that can: (i) suppress anti-MHV68 humoral responses during acute MHV68 infection; and (ii) plays a critical role in the observed suppression of anti-malarial humoral responses in the setting of co-infection. Notably, co-infection with an M2-null mutant MHV68 eliminates lethality of <i>P</i>. <i>yoelii</i> XNL. Collectively, our data demonstrates that an acute gammaherpesvirus infection can negatively impact the development of an anti-malarial immune response. This suggests that acute infection with EBV should be investigated as a risk factor for non-cerebral severe malaria in young children living in areas endemic for <i>Plasmodium</i> transmission.</p></div

Acute, but not latent, MHV68 infection results in suppressed humoral response.

<p>(A) Timeline of infection. C57BL/6 mice were infected with 1000 PFU of MHV68 IN at day -60, -30, -15 or -7 and challenged with 10⁵ pRBCs on day 0. Absolute number of (B) splenic GC B cell (B220+ GL7+ CD95+) and plasma cell (CD3- B220int CD138+) populations at day 16 post <i>P</i>. <i>yoelii</i> XNL infection (For GC and PC: Day -7 and Day -15 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ Day -30 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (C) MHV68 and <i>P</i>. <i>yoelii</i> XNL specific IgG responses at day 16 post <i>P</i>. <i>yoelii</i> XNL infection (Day -7 and Day -15 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ Day -30 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (D) Global Tfh population (CD4+ PD-1+ CXCR5+), germinal center Tfh (CD4+ GL7+ CXCR5+) and activated/antigen specific Tfh (CD4+ CD44+ PD-1+ CXCR5+) in the spleen at day 16 post <i>P</i>. <i>yoelii</i> XNL infection.</p

MHV68 and <i>Plasmodium</i> co-infection results in defective splenic T follicular helper (Tfh) response.

<p>The timeline and experimental set up was identical to that shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004858#ppat.1004858.g001" target="_blank">Fig 1A</a>. (A) Representative flow plots for gating strategies used to define the global Tfh population (CD4+ PD-1+ CXCR5+), germinal center Tfh (CD4+ GL7+ CXCR5+) and activated/antigen specific Tfh (CD4+ CD44+ PD-1+ CXCR5+). (B) Absolute values for all three Tfh subsets are plotted for the <i>P</i>. <i>yoelii</i> XNL (Day 23, all Tfh subsets, <i>P</i>. <i>yoelii</i> vs. co-infected, p<0.05 Mann Whitney U-test) or (C) <i>P</i>. <i>chabaudi</i> co-infection models at multiple time points (Day 23, all Tfh subsets, <i>P</i>. <i>chabaudi</i> vs. co-infected, p<0.05 Mann Whitney U-test).</p