Gamma-herpesvirus colonization of the spleen requires lytic replication in B cells

Gamma-herpesviruses infect lymphocytes and cause lymphocytic cancers. Murid Herpesvirus-4 (MuHV-4), Epstein-Barr virus and the Kaposi's Sarcoma-associated Herpesvirus all infect B cells. Latent infection can spread by B cell recirculation and proliferation, but whether this alone achieves systemic infection is unclear. To test the need of MuHV-4 for lytic infection in B cells we flanked its essential ORF50 lytic transactivator with loxP sites, then infected mice with B cell-specific cre expression (CD19-cre). The floxed virus replicated normally in cre- mice. In CD19-cre mice, nasal and lymph node infections were maintained but there was little splenomegaly and splenic virus loads remained low. Cre-mediated removal of other essential lytic genes gave a similar phenotype. CD19-cre spleen infection by intraperitoneal virus was also impaired. Therefore MuHV-4 had to emerge lytically from B cells to colonize the spleen. An important role for B cell lytic infection in host colonization is consistent with the large CD8+ T cell responses made to gamma-herpesvirus lytic antigens during infectious mononucleosis, and suggests that vaccine-induced immunity capable of suppressing B cell lytic infection might reduce long-term virus loads.IMPORTANCE Gamma-herpesviruses cause B cell cancers. Most models of host colonization derive from cell cultures with continuous, virus-driven B cell proliferation. However vaccines based on these models have worked poorly. To test whether proliferating B cells suffice for host colonization, we inactivated the capacity of MuHV-4, a gamma-herpesvirus of mice, to re-emerge from B cells. The modified virus was able to colonize a first wave of B cells in lymph nodes, but spread poorly to B cells in secondary sites such as the spleen. Consequently viral loads remained low. These results were consistent with virus-driven B cell proliferation exploiting normal host pathways, and so having to transfer lytically to new B cells for new proliferation. We conclude that viral lytic infection is a potential target to reduce B cell proliferation

Murine Cytomegalovirus glycoprotein O promotes epithelial cell infection in vivo

Cytomegaloviruses (CMVs) establish systemic infections across diverse cell types. Glycoproteins that alter tropism can potentially guide their spread. Glycoprotein O (gO) is a non-essential fusion complex component of both human (HCMV) and murine CMV (MCMV). We tested its contribution to MCMV spread from the respiratory tract. , MCMV lacking gO poorly infected fibroblasts and epithelial cells. Cell binding was intact but penetration was delayed. By contrast myeloid infection was preserved, and in the lungs, where myeloid and type 2 alveolar epithelial cells are the main viral targets, MCMV lacking gO showed a marked preference for myeloid infection. Its poor epithelial cell infection was associated with poor primary virus production and reduced virulence. Systemic spread, which proceeds via infected CD11c myeloid cells, was initially intact but then diminished, because less epithelial infection led ultimately to less myeloid infection. Thus, tight linkage between peripheral and systemic MCMV infections gave gO-dependent infection a central role in host colonization. Human cytomegalovirus is a leading cause of congenital disease. This reflects its capacity for systemic spread. A vaccine is needed, but the best viral targets are unclear. Attention has focussed on the virion membrane fusion complex. It has 2 forms, so we need to know what each contributes to host colonization. One includes the virion glycoprotein O. We used murine cytomegalovirus, which has equivalent fusion complexes, to determine the importance of glycoprotein O after mucosal infection. We show that it drives local virus replication in epithelial cells. It was not required to infect myeloid cells, which establish systemic infection, but poor local replication reduced systemic spread as a secondary effect. Therefore targeting glycoprotein O of human cytomegalovirus has the potential to reduce both local and systemic infections

Murine cytomegalovirus degrades MHC class II to colonize the salivary glands

<div><p>Cytomegaloviruses (CMVs) persistently and systemically infect the myeloid cells of immunocompetent hosts. Persistence implies immune evasion, and CMVs evade CD8⁺ T cells by inhibiting MHC class I-restricted antigen presentation. Myeloid cells can also interact with CD4⁺ T cells via MHC class II (MHC II). Human CMV (HCMV) attacks the MHC II presentation pathway <i>in vitro</i>, but what role this evasion might play in host colonization is unknown. We show that Murine CMV (MCMV) down-regulates MHC II via M78, a multi-membrane spanning viral protein that captured MHC II from the cell surface and was necessary although not sufficient for its degradation in low pH endosomes. M78-deficient MCMV down-regulated MHC I but not MHC II. After intranasal inoculation, it showed a severe defect in salivary gland colonization that was associated with increased MHC II expression on infected cells, and was significantly rescued by CD4⁺ T cell loss. Therefore MCMV requires CD4⁺ T cell evasion by M78 to colonize the salivary glands, its main site of long-term shedding.</p></div

M78 relocalizes MHC II.

<p><b>a</b>. Cloned RAW-C2TA cells were transduced with vector alone or with M78 (line 1 and line 2), or infected with MCMV-GFP (+ MCMV, 1 p.f.u. / cell, 72h), then stained for MHC II (dashed lines). Solid lines show uninfected, untransduced RAW-C2TA cells. The biphasic population with MCMV corresponds to GFP⁺ (MHC II^-) and GFP^- (MHC II⁺) cells. M78 transfection had much less effect than MCMV infection on cell surface MHC II expression. <b>b</b>. MCMV-GFP-infected RAW-C2TA cells were stained for M78. GFP was visualized directly. Nuclei were stained with DAPI. Arrows show example infected cells with M78 in vesicles. <b>c</b>. Untransduced (control) and M78-transduced RAW-C2TA cells were stained for M78 and CD44. Arrows show M78 staining, which did not co-localize with CD44. <b>d</b>. M78-transduced RAW-C2TA cells were stained for M78 and MHC II. M78 occupied intracellular vesicles, as did MHC class II in M78⁺ cells. White arrows show examples of co-localization. Grey arrows show M78^- cells with MHC II on the plasma membrane. <b>e</b>. Macrophages grown from bone marrow were infected with WT MCMV (1 p.f.u. / cell, 24h), then fixed and stained for M78 and MHC II. The white arrow shows a typical uninfected MHC II⁺ cell. The yellow arrow shows a typical infected cell with MHC II in vesicles. >80% of vesicles were M78⁺MHC II⁺, <20% were M78⁺MHC II^- and <1% were M78^-MHC II⁺ (n>300). Thus there was significant endosomal M78 / MHC II co-localization (p<10^-4 by Fisher's exact test). <b>f</b>. RAW-C2TA cells were transfected with a plasmid expressing HA-tagged M78. 3d later they were fixed, permeabilized and stained for M78 and MHC II. Nuclei were stained with DAPI. Arrows show examples of M78 and MHC II co-localizing in internal vesicles. <b>g</b>. Cloned RAW-C2TA-M78 cells were stained for MHC II and M78. White arrows show M78⁺ cells with MHC II in vesicles; grey arrows show cells with diffuse M78 staining and MHC II not relocalized. M78/MHC II co-localization was observed in >30% of transduced cells (n>500) and not in untransduced controls. <b>h</b>. RAW-C2TA cells were infected with HA-M78⁺ MCMV (4h, 37°C), incubated with rabbit anti-HA and rat anti-MHC II IgG (1h, 4°C), then washed and incubated at 37°C for the times indicated before fixation, permeabilization and staining with anti-rat (red) and anti-rabbit (green) antibodies. Nuclei were stained with DAPI. Stainings show the change in distribution of surface-labelled M78 from 0 to 30min, and at 30min M78 co-localization with internalized MHC II (yellow, arrows). <b>i</b>. Summary of staining results from <b>h</b> shows similar kinetics of surface-labelled M78 and MHC II loss. Cells were scored as stained or not regardless of internalization. Each point shows mean ± SEM of 5 fields of view and >100 cells counted. <b>j</b>. RAW-C2TA cells were transfected with plasmid expressing HA-tagged M78, or HA-tagged CCR5 as a control. 3d later they were incubated with rabbit anti-HA IgG (1h, 4°C), then washed and either fixed and permeabilized at once (0 min) or first incubated at 37°C (60 min). All cells were then stained with for rabbit IgG (green). Nuclei were stained with DAPI. HA-CCR5 detection was maintained over 60min, whereas HA-M78 detection was lost.</p

Significant M78^- MCMV rescue by CD4⁺ T cell loss.

<p><b>a</b>. C57BL/6 mice were given WT or M78^- MCMV i.n. (3x10⁴ p.f.u.). 56d later sera were assayed for MCMV-specific IgG and IgM by ELISA. Naive = age-matched, uninfected controls. Each point shows the mean of results for 7 mice. M78^- MCMV elicited significantly less IgG response than WT (p<0.01). <b>b</b>. C57BL/6 mice were given WT or M78^- MCMV, or as a control MuHV-4 i.n. (3x10⁴ p.f.u.). 56d after MCMV infection or 10d after MuHV-4 infection, CD4⁺ T cells were purified from splenocytes, pooled from 2 mice per group, by depleting other cells with magnetic beads (Untouched mouse CD4 cell kit, Thermofisher). IFNγ production in response to MCMV-exposed or MuHV-4-exposed naive syngeneic spleen cells (1 p.f.u. / cell) was measured by ELIspot assay. Symbols show replicate wells, bars show means. <b>c</b>. C57BL/6 mice were given WT or M78^- MCMV i.n. (3x10⁴ p.f.u.). 56d later IFNγ production by splenocytes exposed to uninfected or MCMV-exposed naive syngeneic spleen cells was measured by ELIspot assay. Symbols show individual mice, bars show means. CD4⁺ T cell responses to WT and M78^- MCMV were not significantly different. <b>d</b>. BALB/c mice were depleted of CD4⁺ or CD8⁺ T cells (αCD4, αCD8) or left undepleted (cont), then given i.n. WT or M78^- MCMV (3x10⁴ p.f.u.). 10d later lungs and SG were plaque assayed for infectious virus. Symbols show individuals, bars show means. In lungs, immune depletions did not significantly change the ratio of WT to M78^- titers. In SG, CD4⁺ T cell depletion significantly reduced this ratio. <b>e</b>. Viral DNA loads of SG in <b>d</b> were determined by QPCR. Again CD4⁺ T cell depletion significantly reduced the ratio of WT to M78^- infection, that is significantly reversed the M78^- infection defect. <b>f</b>. Immunocompetent (IA^+/-) and MHC II^- (IA^-/-) C57BL/6 mice were given WT, M78^- or revertant (REV) MCMV i.n. (3x10⁴ p.f.u.). At d10 lungs and SG were plaque-assayed for infectious virus. Symbols show individual mice, bars show means. For both lungs (p<0.05) and SG (p<0.01), CD4⁺ T cell loss (IA^-/- mice) significantly increased M78^- plaque titers relative to WT or REV. <b>g</b>. For the mice in <b>f</b>, CD4⁺ T cell loss significantly increased M78^- viral DNA loads in SG relative to WT or REV (p<0.05). <b>h</b>. IA^-/- and IA^+/- mice were given i.n. WT, M131^- or M78 deletion mutant (M78^-I) MCMV. At d10 lungs and SG were plaque assayed for infectious virus. Symbols show individuals, bars show means. The ratio of M78^-I/WT salivary gland titers was significantly higher in IA^-/- than IA^+/- mice (p<0.01), while the ratio of M131^-/WT salivary gland titers was reduced. <b>i</b>. BALB/c mice were depleted of CD4⁺ T cells as in <b>d</b>, then given WT or M33^- MCMV i.n.. After 10d SG were plaque assayed for infectious virus. Symbols show individuals, bars show means. CD4⁺ T cell depletion increased WT MCMV SG infection but failed to rescue SG infection by M33^- MCMV. <b>j</b>. BALB/c mice were depleted of CD4⁺ T cells and given WT or M78^- MCMV i.n. as in <b>d</b>. At d10 salivary gland sections were stained for MHC II and MCMV IE1. UI = uninfected control. Arrows show example infected cells (speckled nuclear IE1 staining). These cells were all MHC II^- with WT MCMV and MHC II⁺ with M78^- MCMV. Images are representative of 6 mice per group.</p

MCMV degrades MHC II.

<p><b>a</b>. RAW-C2TA cells infected with MCMV-GFP (0.5 p.f.u. / cell, 48h) were analysed for surface MHC II by flow cytometry. Mean fluorescence intensity was >10-fold lower on GFP⁺ cells than on GFP^-. CD44 and CD71 mean fluorescence intensity of GFP⁺ cells was reduced <2-fold. Numbers show % cells in each quadrant. Each data set represents at least 3 experiments. <b>b</b>. RAW-C2TA cells were uninfected or infected with MCMV-βgal (1 p.f.u. / cell, 72h), then fixed, permeabilized and stained for βgal and MHC II. White arrows show example MHC II^- infected cells; dark grey arrows show MHC II⁺ uninfected cells in the same cultures; light grey arrows show weakly βgal⁺ cells with MHC II in internal vesicles. Significantly fewer MCMV⁺ cells (<10%, n>200) than MCMV^- cells (>90%, n>200) were MHC II⁺ (p<10^-4 by Fisher's exact test). >100 weakly βgal⁺ cells showed MHC II redistribution. <b>c</b>. RAW-C2TA cells were infected as in <b>b</b>. MHC II⁺ cells were identified with conformation-dependent (M5/114) and -independent (IBL5/22) antibodies. Arrows show example infected cells. Significantly fewer GFP⁺ (<15%, n>100) than GFP^- cells (>90%, n>100) were M5/114⁺ and IBL5/22⁺ (p<10^-4 by Fisher's exact test). <b>d</b>. Peritoneal macrophages were recovered from BALB/c or C57BL/6 mice 72h after i.p. thiglycollate. Non-adherent cells were discarded. The remaining cells were infected or not with MCMV-GFP (1 p.f.u. / cell, based on total cell numbers before adherence). 48h later surface MHC II and CD44 were analysed by flow cytometry. Numbers show % total cells in each quadrant, collecting >5000 cells. In uninfected cultures, macrophages were 22% (BALB/c) and 26% (C57BL/6) MHC II⁺. In infected cultures, GFP^- macrophages were 27% (BALB/c) and 24% (C57BL/6) MHC II⁺, while GFP⁺ macrophages were 0.3% (BALB/c) and 0.2% (C57BL/6) MHC II⁺ (p<10^-4 by X² test).</p

M78^- MCMV replication <i>in vivo</i>.

<p><b>a</b>. BALB/c mice were given i.n. WT or M78^- MCMV (3x10⁴ p.f.u.). 3d later lung sections were stained for MCMV IE1 and MHC II. Nuclei were stained with DAPI. Arrows show infected cells. <b>b</b>. Quantitation of staining as in <b>a</b>, for sections from 3 mice. Few WT infected cells were MHC II⁺. At d1 and d3, significantly more M78^--infected cells were MHC II⁺. <b>c</b>. Mice were infected as in <b>a</b>. Lungs and salivary glands were plaque-assayed for infectious virus, and QPCR-assayed for viral genomes relative to cellular (βactin) genomes. Bars show means, other symbols show individual mice. Dashed lines show assay sensitivity limits. Significant differences are indicated.</p

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.Originally included in thesis in manuscript form. </p