TNF Signaling Dictates Myeloid and Non-Myeloid Cell Crosstalk to Execute MCMV-Induced Extrinsic Apoptosis.

Cytomegaloviruses all encode the viral inhibitor of caspase-8-induced apoptosis (vICA). After binding to this initiator caspase, vICA blocks caspase-8 proteolytic activity and ability to activate caspase-3 and/or caspase-7. In this manner, vICA has long been known to prevent apoptosis triggered via tumor necrosis factor (TNF) family death receptor-dependent extrinsic signaling. Here, we employ fully wild-type murine cytomegalovirus (MCMV) and vICA-deficient MCMV (∆M36) to investigate the contribution of TNF signaling to apoptosis during infection of different cell types. ∆M36 shows the expected ability to kill mouse splenic hematopoietic cells, bone marrow-derived macrophages (BMDM), and dendritic cells (BMDC). Antibody blockade or genetic elimination of TNF protects myeloid cells from death, and caspase-8 activation accompanies cell death. Interferons, necroptosis, and pyroptotic gasdermin D (GSDMD) do not contribute to myeloid cell death. Human and murine fibroblasts or murine endothelial cells (SVEC4-10) normally insensitive to TNF become sensitized to ∆M36-induced apoptosis when treated with TNF or TNF-containing BMDM-conditioned medium. We demonstrate that myeloid cells are the natural source of TNF that triggers apoptosis in either myeloid (autocrine) or non-myeloid cells (paracrine) during ∆M36 infection of mice. Caspase-8 suppression by vICA emerges as key to subverting innate immune elimination of a wide variety of infected cell types.

Cytomegalovirus vaccine strain towne-derived dense bodies induce broad cellular immune responses and neutralizing antibodies that prevent infection of fibroblasts and epithelial cells.

Human cytomegalovirus (HCMV), a betaherpesvirus, can cause severe disease in immunosuppressed patients and following congenital infection. A vaccine that induces both humoral and cellular immunity may be required to prevent congenital infection. Dense bodies (DBs) are complex, noninfectious particles produced by HCMV-infected cells and may represent a vaccine option. As knowledge of the antigenicity and immunogenicity of DB is incomplete, we explored characterization methods and defined DB production methods, followed by systematic evaluation of neutralization and cell-mediated immune responses to the DB material in BALB/c mice. DBs purified from Towne-infected cultures treated with the viral terminase inhibitor 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) were characterized by nanoparticle tracking analysis (NTA), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), immunoblotting, quantitative enzyme-linked immunosorbent assay, and other methods. The humoral and cellular immune responses to DBs were compared to the immunogenicity of glycoprotein B (gB) administered with the adjuvant AddaVax (gB/AddaVax). DBs induced neutralizing antibodies that prevented viral infection of cultured fibroblasts and epithelial cells and robust cell-mediated immune responses to multiple viral proteins, including pp65, gB, and UL48. In contrast, gB/AddaVax failed to induce neutralizing antibodies that prevented infection of epithelial cells, highlighting a critical difference in the humoral responses induced by these vaccine candidates. Our data advance the potential for the DB vaccine approach, demonstrate important immunogenicity properties, and strongly support the further evaluation of DBs as a CMV vaccine candidate.

Gene products of the embedded m41/m41.1 locus of murine cytomegalovirus differentially influence replication and pathogenesis.

Cytomegaloviruses utilize overlapping and embedded reading frames as a way to efficiently package and express all genes necessary to carry out a complex lifecycle. Murine cytomegalovirus encodes a mitochondrial-localized inhibitor of Bak oligomerization (vIBO) from m41.1, a reading frame that is embedded within the m41 gene. The m41.1-encoded mitochondrial protein and m41-encoded Golgi-localized protein have both been implicated in cell death suppression; however, their contribution to viral infection within the host has not been investigated. Here, we report that mitochondrial-localized m41.1 (vIBO) is required for optimal viral replication in macrophages and has a modest impact on dissemination in infected mice. In contrast, Golgi-localized m41 protein is dispensable during acute infection and dissemination as well as for latency. All together, these data indicate that the primary evolutionary focus of this locus is to maintain mitochondrial function through inhibition of Bak-mediated death pathways in support of viral pathogenesis.

Antiviral T cell response triggers cytomegalovirus hepatitis in mice.

One common sign of human cytomegalovirus infection is altered liver function. Murine cytomegalovirus strain v70 induces a rapid and severe hepatitis in immunocompetent mice that requires the presence of T cells in order to develop. v70 exhibits approximately 10-fold-greater virulence than the commonly used strain K181, resulting in a more severe, sustained, and lethal hepatitis but not dramatically higher viral replication levels. Hepatitis and death are markedly delayed in immunodeficient SCID compared to immunocompetent BALB/c mice. Transfer of BALB/c splenocytes to SCID mice conferred rapid disease following infection, and depletion of either CD4 or CD8 T cells in BALB/c mice reduced virus-induced hepatitis. The frequency of CD8 T cells producing gamma interferon and tumor necrosis factor in response to viral antigen was higher in settings where more severe disease occurred. Thus, virus-specific effector CD8 T cells appear to contribute to lethal virus-induced hepatitis, contrasting their protective role during sublethal infection. This study reveals how protection and disease during cytomegalovirus infection depend on viral strain and dose, as well as the quality of the T cell response.

The human cytomegalovirus UL36 gene controls caspase-dependent and -independent cell death programs activated by infection of monocytes differentiating to macrophages.

The cellular protease caspase-8 activates extrinsic apoptosis and also functions to promote monocyte-to-macrophage differentiation. Differentiation-induced alterations to antiviral caspase-8-dependent cell death pathways are unclear. Here, we show THP-1 monocyte-to-macrophage differentiation alters the specific cell death pathways activated in response to human cytomegalovirus (HCMV) infection. Employing viruses with mutations in UL36, the gene that encodes the viral inhibitor of caspase-8 activation (vICA), our data indicate that both caspase-dependent and -independent death pathways are activated in response to infection. Activation of caspase-dependent and -independent cell death responses restricted growth of vICA-deficient viruses, and vICA/pUL36 inhibited either response. Thus, these studies also reveal that the UL36 gene controls a caspase-independent cell death pathway. The impact of caspases on control of antiviral responses differed at early and late stages of macrophage differentiation. Early in differentiation, vICA-deficient virus-induced cell death was dependent on caspases and inhibited by the pan-caspase inhibitor z-VAD(OMe)-fluoromethyl ketone. In contrast, virus-induced death at late times of differentiation was caspase independent. Additional unlabeled and fluorescent inhibitors indicated that caspase-8 promoted death from within infected cells at early but not late stages of differentiation. These data highlight the multifunctional role of vICA/pUL36 as HCMV encounters various antiviral responses during macrophage differentiation.

Frequent occult infection with Cytomegalovirus in cardiac transplant recipients despite antiviral prophylaxis.

Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D- and R+/D+) experienced a greater infection burden than the R-/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (< 3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10(5) polymorphonuclear leukocytes) than the R-/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.

Disruption of mitochondrial networks by the human cytomegalovirus UL37 gene product viral mitochondrion-localized inhibitor of apoptosis.

By 24 h after infection with human cytomegalovirus, the reticular mitochondrial network characteristic of uninfected fibroblasts was disrupted as mitochondria became punctate and dispersed. These alterations were associated with expression of the immediate-early (alpha) antiapoptotic UL37x1 gene product viral mitochondrion-localized inhibitor of apoptosis (vMIA). Similar alterations in mitochondrial morphology were induced directly by vMIA in transfected cells. A 68-amino-acid antiapoptotic derivative of vMIA containing the mitochondrial localization and antiapoptotic domains also induced disruption, whereas a mutant lacking the antiapoptotic domain failed to cause disruption. These data suggest that the fission and/or fusion process that normally controls mitochondrial networks is altered by vMIA. Mitochondrial fission has been implicated in the induction of apoptosis and vMIA-mediated inhibition of apoptosis may occur subsequent to this event.