Bahram Ebrahimi

Murine gammaherpesvirus-68 infection causes multi-organ fibrosis and alters leukocyte trafficking in interferon-γ receptor knockout mice

Murine gammaherpesvirus-68 (MHV-68) infection in interferon-gamma receptor knockout mice (IFN-gammaR(-)/(-)) results in splenic fibrosis and excessive loss of splenocytes. In our present study we found that MHV-68 infection in IFN-gammaR(-)/(-) mice also resulted in fibrosis and atrophy of the mediastinal lymph nodes, interstitial pulmonary fibrosis and fibrotic changes in the liver. Atrophy and cellular depletion of the spleen in IFN-gammaR(-)/(-) was not the result of increased cell death. The loss of splenocytes in IFN-gammaR(-)/(-) mice, which was most evident on day 23 after infection, correlated with an increase in the number of leukocytes in peripheral blood. At the peak of leukocytosis, on day 23 after infection, peripheral blood cells from infected IFN-gammaR(-)/(-) mice were unable to traffic through the fibrosed spleens of IFN-gammaR(-)/(-) mice but were able to enter the spleens of wild-type mice. This indicates that leukocytosis was in part the result of emigration of cells from the spleen and their subsequent exclusion of re-entry at the height of fibrosis. Significant cytokine and chemokine changes were observed in spleens of IFN-gammaR(-)/(-) mice. IFN-gamma, tumor necrosis factor-alpha (TNF-alpha ), TNF-beta, interleukin-1beta (IL-1beta), transforming growth factor-beta1 (TGF-beta1), lymphotactin, and MIP-1beta were elevated on day 14 after infection whereas chemokines IP-10 and MIG were significantly reduced. These changes suggest a role for dysregulated cytokines and chemokines in severe organ-specific fibrosis with implications for immune-mediated fibrotic disorders.

Murine gammaherpesvirus M11 gene product inhibits apoptosis and is expressed during virus persistence

Summary. The murine gammaherpesvirus (MHV-68) M11 gene encodes a protein with BH1 domain homology to Bcl-2. We found that the M11 gene product (MHVBcl-2) protected murine epithelial cells from TNF-α induced apoptosis. M11 was transcribed during early lytic infection in vitro. During early infection of mice, M11 message was detected in spleen and lung along with lytic cycle messages. During persistence, lytic cycle gene expression was undetectable but M11 RNA was still present. This suggests that MHVBcl-2 promotes virus survival by protecting not only productively infected but also persistently infected cells from apoptotic death.

Severe Respiratory Syncytial Virus Bronchiolitis in Infants Is Associated with Reduced Airway Interferon Gamma and Substance P

Background Severe human respiratory syncytial virus (hRSV) bronchiolitis in previously well infants may be due to differences in the innate immune response to hRSV infection. Aim: to determine if factors mediating proposed mechanisms for severe bronchiolitis differ with severity of disease. Methodology/Principle Findings 197 infants admitted to hospital with hRSV bronchiolitis were recruited and grouped according to no oxygen requirement (n = 27), oxygen dependence (n = 114) or mechanical ventilation (n = 56). We collected clinical data, nasopharyngeal aspirate (NPA) and if ventilated bronchoalveolar lavage (BAL). Interferon-gamma (IFN-γ), substance P (SP), interleukin 9 (IL-9), urea and hRSV load, were measured in cell free supernatant from NPA and BAL. Multivariate analysis compared independent effects of clinical, virological and immunological variables upon disease severity. IFN-γ and SP concentrations were lower in NPA from infants who required oxygen or mechanical ventilation. Viral load and IL-9 concentrations were high but did not vary with severity of disease. Independent predictors of severe disease (in diminishing size of effect) were low weight on admission, low gestation at birth, low NPA IFN-γ and NPA SP. Nasal airway sampling appears to be a useful surrogate for distal airway sampling since concentrations of IFN-γ, SP, IL-9 and viral load in NPA correlate with the same in BAL. Conclusions Our data support two proposed mechanisms for severe hRSV disease; reduced local IFN-γ response and SP mediated inflammation. We found large amounts of hRSV and IL-9 in airways secretions from the upper and lower respiratory tract but could not associate these with disease severity.

Murine gammaherpesvirus-induced fibrosis is associated with the development of alternatively activated macrophages

Murine gammaherpesvirus 68 (MHV‐68) is a natural pathogen of rodents closely related to the human γherpesviruses Kaposi’s sarcoma‐associated herpesvirus and EBV. Following intranasal infection, the virus replicates in the lung epithelium prior to establishing latent infection in lymphoid tissue. Infection of mice deficient in IFN‐γR signaling (IFN‐γR−/−) results in a multiple organ fibrosis, in which the spleen is severely affected. We show here that by Day 12 postinfection, prior to development of fibrosis in the spleens of IFN‐γR−/− mice, different subsets of splenic macrophages (Mϕs) are morphologically activated and enter latently infected germinal centers (GCs). Mϕs coexpressing arginase I (ARG1), a marker of alternative activation of Mϕs, and murine Mϕ markers F4/80, ER‐TR9, and MOMA‐1 are found in GCs of IFN‐γR−/− mice but not of wild‐type mice. Quantitative RT‐PCR of spleen RNA confirms induction of ARG1 and in addition, shows up‐regulation of found in inflammatory zone 1/resistin‐like molecule‐α, tissue inhibitor of metalloproteinase‐1, matrix metalloproteinase‐12, fibronectin, and factor XIIIA in IFN‐γR−/− mice. In contrast, inducible NO synthase, associated with classical Mϕ activation, is up‐regulated following infection of wild‐type mice but not IFN‐γR−/− mice. Concomitant with the aaMϕs, transcription of the Th2 cytokines IL‐13, IL‐21, and IL‐5 is up‐regulated. Thus, in the absence of IFN‐γR signaling, MHV‐68 initiates a Th2 immune response, leading to alternative activation of macrophages and induction of fibrosis. This system provides an important model for studying the pathogenesis of fibrosis initiated by a latent herpesvirus infection.

Regulation of ecdysteroid signalling during Drosophila development : identification, characterization and modelling of ecdysone oxidase, an enzyme involved in control of ligand concentration

The steroidal moulting hormones (ecdysteroids) mediate developmental transitions in insects, and their regulation is mainly controlled by the production and inactivation of these steroid hormones at the appropriate developmental times. One route of metabolism of ecdysteroids in insects involves EO (ecdysone oxidase)-catalysed conversion into 3-dehydroecdysteroid, which undergoes reduction to the corresponding 3-epiecdysteroid. By a twin-stranded bioinformatics approach, employing both phylogenomics and model structure-based analysis, we first predicted that DmEO (the EO of Drosophila melanogaster) corresponds to the protein product of gene CG9504. When CG9504 was expressed in COS7 cells, significant conversion of ecdysone into 3-dehydroecdysone was observed. Quantitative PCR and enzyme assay showed that DmEO was mainly expressed in the midgut during the late instars at a time corresponding to a hormone titre peak. DmEO shares only 27% amino acid sequence identity with Spodoptera littoralis (Lepidoptera) EO, yet key substrate-binding residues are well conserved. A model of DmEO is consistent with an inability to catalyse reaction of cholesterol derivatives. The significance of DmEO in ligand activation is discussed in relation to new evidence suggesting that 3-dehydro- and 3-epiecdysteroids may be functionally active as ligands in a novel, atypical ecdysteroid signalling pathway involving the Drosophila orphan nuclear receptor, DHR38, rather than being merely hormone inactivation products.

Crystal structure of a KSHV–SOX–DNA complex: insights into the molecular mechanisms underlying DNase activity and host shutoff

The early lytic phase of Kaposi’s sarcoma herpesvirus infection is characterized by viral replication and the global degradation (shutoff) of host mRNA. Key to both activities is the virally encoded alkaline exonuclease KSHV SOX. While the DNase activity of KSHV SOX is required for the resolution of viral genomic DNA as a precursor to encapsidation, its exact involvement in host shutoff remains to be determined. We present the first crystal structure of a KSHV SOX–DNA complex that has illuminated the catalytic mechanism underpinning both its endo and exonuclease activities. We further illustrate that KSHV SOX, similar to its Epstein–Barr virus homologue, has an intrinsic RNase activity in vitro that although an element of host shutoff, cannot solely account for the phenomenon.

In Vivo Function of a Gammaherpesvirus Virion Glycoprotein: Influence on B-Cell Infection and Mononucleosis

ABSTRACT The human gammaherpesviruses Epstein-Barr virus and Kaposi Sarcoma-associated herpesvirus both contain a glycoprotein (gp350/220 and K8.1, respectively) that mediates binding to target cells and has been studied in great detail in vitro. However, there is no direct information on the role that these glycoproteins play in pathogenesis in vivo. Infection of mice by murid herpesvirus 4 strain 68 (MHV-68) is an established animal model for gammaherpesvirus pathogenesis and expresses an analogous glycoprotein, gp150. To elucidate the in vivo function of gp150, a recombinant MHV-68 deficient in gp150 production was generated (vgp150Δ). The productive viral replication in vitro and in vivo was largely unaffected by mutation of gp150, aside from a partial defect in the release of extracellular virus. Likewise, B-cell latency was established. However, the transient mononucleosis and spike in latently infected cells associated with the spread of MHV-68 to the spleen was significantly reduced in vgp150Δ-infected mice. A soluble, recombinant gp150 was found to bind specifically to B cells but not to epithelial cells in culture. In addition, gp150-deficient MHV-68 derived from mouse lungs bound less well to spleen cells than wild-type virus. Thus, gp150 is highly similar in function in vitro to the Epstein-Barr virus gp350/220. These results suggest a role for these analogous proteins in mononucleosis and have implications for their use as vaccine antigens.

Human cytomegalovirus transcriptome activity differs during replication in human fibroblast, epithelial and astrocyte cell lines.

Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed.

A shutoff and exonuclease mutant of murine gammaherpesvirus-68 yields infectious virus and causes RNA loss in type I interferon receptor knockout cells

Significant loss of RNA followed by severely reduced cellular protein pool, a phenomenon termed host shutoff, is associated with a number of lytic virus infections and is a critical player in viral pathogenesis. Until recently, viral DNA exonucleases were associated only with processing of viral genomic DNA and its encapsidation. However, recent observations have identified host shutoff and exonuclease function for the highly conserved viral exonucleases in γ-herpesviruses, which include Kaposis sarcoma-associated herpesvirus, Epstein-Barr virus and the mouse model murine gammaherpesvirus-68, also referred to as MHV-68. In this study, we show that although ablation of the MHV-68 exonuclease ORF37 caused a restrictive phenotype in WT IFN-α/β receptor-positive cells such as NIH 3T3, lack of ORF37 was tolerated in cells lacking the IFN-α/β receptor: the ORF37Stop virus was capable of forming infectious particles and caused loss of mRNA in IFN-α/β receptor knockout cells. Moreover, ORF37Stop virus was able to establish lytic infection in the lungs of mice lacking the IFN-α/β receptor. These observations provide evidence that lytic MHV-68 infection and subsequent loss of mRNA can take place independently of ORF37. Moreover, efficient growth of ORF37Stop virus also identifies a role for this family of viral nucleases in providing a window of opportunity for virus growth by overcoming type I IFN-dependent responses.

The gene expression signature associated with TP53 mutation/deletion in chronic lymphocytic leukaemia is dominated by the under-expression of TP53 and other genes on chromosome 17p.

In chronic lymphocytic leukaemia (CLL), TP53 mutation and deletion are strongly associated with one another and with adverse clinical outcome. Mutant TP53 protein typically accumulates to high levels and has been reported to have transcriptional regulatory activity distinct from that of wild‐type TP53. To investigate whether such an effect is relevant to CLL, carefully balanced primary CLL samples with or without TP53 mutation/deletion were compared for their gene expression profiles using high‐density DNA microarrays. Ninety‐six and eight differentially expressed genes were identified, respectively, using two alternative statistical approaches with different stringencies. None of the differentially expressed genes were known to be regulated by mutant TP53, and only four of the 67 under‐expressed genes were known transcriptional targets of wild‐type TP53. Significantly, both approaches showed that gene under‐expression was the dominant feature of TP53‐mutant CLL samples. Furthermore, a disproportionate number of the under‐expressed genes were located on chromosome 17p, the most significant being TP53 itself. Together, these results indicate that any transcriptional regulatory effects of mutant TP53 in CLL cells are overshadowed by the under‐expression of co‐deleted TP53 and other genes on chromosome 17p. Our findings have implications for emerging therapeutic strategies that target mutant TP53.