Immunomodulatory properties of a viral homolog of human interleukin-10 expressed by human cytomegalovirus during the latent phase of infection.

Human cytomegalovirus (HCMV) establishes a latent infection in hematopoietic cells, from which it can reactivate to cause significant disease in immunocompromised individuals. HCMV expresses a functional homolog of the immunosuppressive cytokine interleukin-10 (termed cmvIL-10), and alternate splicing of the cmvIL-10 transcript results in expression of a latency-associated cmvIL-10 transcript (LAcmvIL-10). To determine whether LAcmvIL-10 encodes immunosuppressive functions, recombinant LAcmvIL-10 protein was generated, and its impact on major histocompatibility complex class II (MHC-II) expression was examined on granulocyte macrophage progenitor cells (GM-Ps) and monocytes. LAcmvIL-10 (and cmvIL-10) downregulated MHC-II on the surfaces of both cell types. This downregulation was associated with a decrease in total MHC-II protein and transcription of components of the MHC-II biosynthesis pathway. Unlike cmvIL-10, LAcmvIL-10 did not trigger phosphorylation of Stat3, and its ability to downregulate MHC-II was not blocked by neutralizing antibodies to the human IL-10 receptor, suggesting that LAcmvIL-10 either does not engage the cellular IL-10 receptor or utilizes it in a different manner from cmvIL-10. The impact of LAcmvIL-10 on dendritic cell (DC) maturation was also assessed. In contrast to cmvIL-10, LAcmvIL-10 did not inhibit the expression of costimulatory molecules CD40, CD80, and CD86 and the maturation marker CD83 on DCs, nor did it inhibit proinflammatory cytokines (IL-1alpha, IL-1beta, IL-6 and tumor necrosis factor alpha). Thus, LAcmvIL-10 retains some, but not all, of the immunosuppressive functions of cmvIL-10. As GM-Ps and monocytes support latent infection, expression of LAcmvIL-10 may enable HCMV to avoid immune recognition and clearance during latency.

Effect of phthiocerol dimycocerosate deficiency on the transcriptional response of human macrophages to Mycobacterium tuberculosis.

The control of mycobacterial infections is dependent on the finely tuned synergism between the innate and adaptive immune responses. The macrophage is the major host cell for Mycobacterium tuberculosis and the degree of virulence of mycobacteria may influence the initial macrophage response to infection. The cell wall molecule, phthiocerol dimycocerosate (DIM), is an important virulence factor that influences the early growth of M. tuberculosis in the lungs. To explore the basis for this effect we have compared the early gene response of human THP-1 macrophages to infection with virulent M. tuberculosis and the DIM-deficient DeltafadD26 M. tuberculosis strain using microarrays. Detailed analysis revealed a common core of macrophage genes, which were rapidly induced following infection with both strains, and deficiency of DIM had no significant effect on this initial macrophage transcriptional responses. In addition to chemokines and pro-inflammatory cytokines, the early response genes included components of the Toll-like receptor signalling, antigen presentation and apoptotic pathways, interferon response genes, cell surface receptors and their ligands, including TNF-related apoptosis inducing ligand (TRAIL) and CD40, and other novel genes. Therefore, although fadD26 deficiency is responsible for the early attenuation of the growth of M. tuberculosis in vivo, this effect is not associated with differences in the initial macrophage transcriptional response.

Analysis of immune responses to varicella zoster viral proteins induced by DNA vaccination.

In this study we sought to examine the mechanism by which immune responses were induced following intramuscular injection of mice with DNA expression vectors encoding genes of varicella zoster virus (VZV). Both VZV-specific antibody and T cell proliferative responses were induced by immunization with DNA sequences for the immediate early 62 (IE62) and glycoprotein E (gE). The viral proteins were shown to be expressed in non-regenerating, rather than regenerating muscle cells. After primary immunization, muscle cells did not express major histocompatibility complex (MHC) class II transcripts and little inflammatory response was detected at the site of inoculation. Histochemical staining and non-isotopic in situ hybridization demonstrated that a second injection of IE62 plasmid DNA was again associated with protein synthesis in non-regenerating muscle cells but that a marked inflammatory infiltrate was induced in muscle tissue. These cells, but not muscle cells, expressed MHC class II transcripts. Significantly, PCR analyses demonstrated that IE62 DNA localized specifically to local draining lymph nodes following primary DNA immunization by intramuscular inoculation. These experiments indicate that transport of plasmid DNA to sites of antigen presentation in regional lymphoid tissue may play an important role in the initial generation of immune responses and that enhancement by secondary inoculation is mediated by immune cells that traffic to the site of viral protein synthesis in muscle cells.

Concatemeric intermediates of equine herpesvirus type 1 DNA replication contain frequent inversions of adjacent long segments of the viral genome.

In common with other alpha-herpesviruses, the genome of equine herpesvirus type-1 (EHV-1) comprises covalently linked long and short unique sequences of DNA, each flanked by inverted repeats. Equimolar amounts of two genomic isomers, generated by free inversion of the short segment, relative to the long segment, are packaged into EHV-1 virions. In contrast with herpes simplex virus (HSV), inversion of genomic long segments has not been described. In the current work, the structures of high molecular weight intermediates of EHV-1 DNA replication were studied by field inversion gel electrophoresis. It is shown that adjacent long segments of the viral genome are frequently inverted in concatemeric intermediates of EHV-1 DNA replication. Further, like HSV concatemers, high molecular weight intermediates of EHV-1 replication are flanked exclusively by the long segment of the viral genome. Hence, despite the fact that only two, rather than four, isomers of EHV-1 DNA are packaged into virions, the intermediates of EHV-1 DNA replication closely resemble those of herpes simplex virus type 1 in structure. These data have implications relating to the mechanisms involved in packaging of alpha-herpesvirus DNA.

Quantitative analysis of herpes simplex virus DNA and transcriptional activity in ganglia of mice latently infected with wild-type and thymidine kinase-deficient viral strains.

The relationship between herpes simplex virus (HSV) DNA replication and establishment of latent infection was examined using an experimental model that makes use of the segmental sensory innervation of mouse flanks (T7 to T12). Ganglia from consecutive thoracic segments of C57BL/10 mice latently infected with a virulent strain of HSV-1 (SC16) were compared with respect to (i) HSV DNA levels, (ii) latency-associated transcripts (LATs) and (iii) numbers of LAT+ neurons. In concordance with previous results, two patterns of virus persistence were detected distinguished by either a low (10 to 23) or high (approx. 200) number of viral genomes/LAT+ neuron. The high copy pattern was associated, anatomically, with ganglia directly innervating inoculated skin (T7/8). Paradoxically, the highest number of LAT+ neurons and the highest concentrations of LATs were detected in spinal segments (e.g. T10) containing the lowest number of viral genomes, implying that most of the latent SC16 DNA detected at T7 and T8 was transcriptionally repressed. When neuronal amplification of HSV DNA during the establishment phase was prevented by infecting mice with a viral thymidine kinase deletion mutant (TKDM21), the high copy pattern was eliminated and each LAT+ neuron contained, on average, 22 TKDM21 genomes. We conclude that input (i.e. unamplified) and progeny (i.e. amplified) DNA sequences persist in the peripheral nervous systems of mice infected with SC16. Structurally, latent TKDM21 DNA lacked free genomic termini, consistent with persistence of input DNA in an integrated or circular episomal configuration.

Retention of herpes simplex virus DNA sequences in the nuclei of mouse footpad keratinocytes after recovery from primary infection.

Persistence of herpes simplex virus (HSV) DNA in mouse footpad keratinocytes was studied by non-isotopic in situ hybridization. HSV DNA was retained in keratinocyte nuclei for more than 2 weeks after disappearance of infectious virus and viral antigens. The anatomical location of viral DNA became more superficial with increasing time post-infection, reflecting the migration of cells from the basal layer of the epidermis towards the stratum granulosum. Latency-associated transcripts (LATs) were not detected in footpad cells at any of the times studied. In contrast, LATs were detected readily in the nuclei of lumbar ganglionic neurons innervating HSV DNA positive footpads. It was concluded that, after termination of productive infection in the skin, HSV DNA persists transiently in keratinocyte nuclei, in the absence of abundant latency-associated transcription. An implication of these data is that detection of HSV DNA in the skin may reflect recent, but not necessarily current, cutaneous virus replication.

Herpes simplex virus genome isomerization: origins of adjacent long segments in concatemeric viral DNA.

Herpes simplex virus type 1 DNA isomerization was studied by using a viral mutant, 5B8, lacking the unique SpeI site of its parent, SC16. In coinfected cells, SC16 genomic long segments flanked 5B8 genomes in all possible orientations with similar frequencies. Thus, recombination between progeny of different replication templates is sufficient to explain genomic isomerization.

Quantitative analysis of latent human cytomegalovirus.

Cytomegalovirus latency depends on an interaction with hematopoietic cells in bone marrow and peripheral blood. The distribution of viral DNA was investigated by PCR-driven in situ hybridization (PCR-ISH), and the number of viral genomes per cell was estimated by quantitative competitive PCR during both experimental and natural latent infection. During experimental latent infection of cultured granulocyte-macrophage progenitors, the viral genome was detected in >90% of cells at a copy number of 1 to 8 viral genomes per cell. During natural infection, viral genomes were detected in 0.004 to 0.01% of mononuclear cells from granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow from seropositive donors, at a copy number of 2 to 13 genomes per infected cell. When evaluated by reverse transcription-PCR-ISH, only a small proportion of experimentally infected cells (approximately 2%) had detectable latent transcripts. This investigation identifies the small percentage of bone marrow-derived mononuclear cells that become latently infected during natural infection and suggests that latency may proceed in some cells that fail to encode currently identified latent transcripts.

Human cytomegalovirus latency-associated protein pORF94 is dispensable for productive and latent infection.

Human cytomegalovirus latency in bone marrow-derived myeloid progenitors is characterized by the presence of latency-associated transcripts encoded in the ie1/ie2 region of the viral genome. To assess the role of ORF94 (UL126a), a conserved open reading frame on these transcripts, a recombinant virus (RC2710) unable to express this gene was constructed. This virus replicated at wild-type levels and expressed productive as well as latency-associated ie1/ie2 region transcripts. During latency in granulocyte-macrophage progenitors, RC2710 DNA was detected at levels indistinguishable from wild-type virus, latent-phase transcription was present, and RC2710 reactivated when latently infected cells were cocultured with permissive fibroblasts. These data suggest pORF94 is not required for either productive or latent infection as assayed in cultured cells despite being the only known nuclear latency-associated protein.

Varicella-zoster virus infection of human dendritic cells and transmission to T cells: implications for virus dissemination in the host.

During primary varicella-zoster virus (VZV) infection, it is presumed that virus is transmitted from mucosal sites to regional lymph nodes, where T cells become infected. The cell type responsible for VZV transport from the mucosa to the lymph nodes has not been defined. In this study, we assessed the susceptibility of human monocyte-derived dendritic cells to infection with VZV. Dendritic cells were inoculated with the VZV strain Schenke and assessed by flow cytometry for VZV and dendritic cell (CD1a) antigen expression. In five replicate experiments, 34.4% +/- 6.6% (mean +/- SEM) of CD1a(+) cells were also VZV antigen positive. Dendritic cells were also shown to be susceptible to VZV infection by the detection of immediate-early (IE62), early (ORF29), and late (gC) gene products in CD1a(+) dendritic cells. Infectious virus was recovered from infected dendritic cells, and cell-to-cell contact was required for transmission of virus to permissive fibroblasts. VZV-infected dendritic cells showed no significant decrease in cell viability or evidence of apoptosis and did not exhibit altered cell surface levels of major histocompatibility complex (MHC) class I, MHC class II, CD86, CD40, or CD1a. Significantly, when autologous T lymphocytes were incubated with VZV-infected dendritic cells, VZV antigens were readily detected in CD3(+) T lymphocytes and infectious virus was recovered from these cells. These data provide the first evidence that dendritic cells are permissive to VZV and that dendritic cell infection can lead to transmission of virus to T lymphocytes. These findings have implications for our understanding of how virus may be disseminated during primary VZV infection.

Modulation of major histocompatibility class II protein expression by varicella-zoster virus.

We sought to investigate the effects of varicella-zoster virus (VZV) infection on gamma interferon (IFN-gamma)-stimulated expression of cell surface major histocompatibility complex (MHC) class II molecules on human fibroblasts. IFN-gamma treatment induced cell surface MHC class II expression on 60 to 86% of uninfected cells, compared to 20 to 30% of cells which had been infected with VZV prior to the addition of IFN-gamma. In contrast, cells that were treated with IFN-gamma before VZV infection had profiles of MHC class II expression similar to those of uninfected cell populations. Neither IFN-gamma treatment nor VZV infection affected the expression of transferrin receptor (CD71). In situ and Northern blot hybridization of MHC II (MHC class II DR-alpha) RNA expression in response to IFN-gamma stimulation revealed that MHC class II DR-alpha mRNA accumulated in uninfected cells but not in cells infected with VZV. When skin biopsies of varicella lesions were analyzed by in situ hybridization, MHC class II transcripts were detected in areas around lesions but not in cells that were infected with VZV. VZV infection inhibited the expression of Stat 1alpha and Jak2 proteins but had little effect on Jak1. Analysis of regulatory events in the IFN-gamma signaling pathway showed that VZV infection inhibited transcription of interferon regulatory factor 1 and the MHC class II transactivator. This is the first report that VZV encodes an immunomodulatory function which directly interferes with the IFN-gamma signal transduction via the Jak/Stat pathway and enables the virus to inhibit IFN-gamma induction of cell surface MHC class II expression. This inhibition of MHC class II expression on VZV-infected cells in vivo may transiently protect cells from CD4(+) T-cell immune surveillance, facilitating local virus replication and transmission during the first few days of cutaneous lesion formation.

Two patterns of persistence of herpes simplex virus DNA sequences in the nervous systems of latently infected mice.

The number of herpes simplex virus (HSV) genome equivalents recovered from latently infected mouse spinal ganglia was compared with the proportion of neurons containing latency-associated transcripts (LATs). Two distinct patterns of HSV persistence were observed, depending on the anatomical location of ganglia with respect to the site of cutaneous inoculation. The location of the bulk of latent viral DNA did not correspond with the highest prevalence of LAT+ neurons. Viral DNA was most abundant in spinal ganglia directly innervating the inoculation site and the amount recovered, which was similar to that found previously in human trigeminal ganglia, suggested that LAT+ neurons each contain hundreds of copies of HSV DNA. In stark contrast, although LAT+ neurons were most abundant in neighbouring ganglia, viral DNA was scarce (approx. 20 copies/LAT+ cell). These data indicate that amplification of HSV DNA sequences is greatest in ganglia previously shown to be associated with viral antigen expression during the productive phase of primary infection.

Varicella-zoster virus retains major histocompatibility complex class I proteins in the Golgi compartment of infected cells.

We sought to examine the effects of varicella-zoster virus (VZV) infection on the expression of major histocompatibility complex class I (MHC I) molecules by human fibroblasts and T lymphocytes. By flow cytometry, VZV infection reduced the cell surface expression of MHC I molecules on fibroblasts significantly, yet the expression of transferrin receptor was not affected. Importantly, when human fetal thymus/liver implants in SCID-hu mice were inoculated with VZV, cell surface MHC I expression was downregulated specifically on VZV-infected human CD3+ T lymphocytes, a prominent target that sustains VZV viremia. The stage in the MHC I assembly process that was disrupted by VZV in fibroblasts was examined in pulse-chase and immunoprecipitation experiments in the presence of endoglycosidase H. MHC I complexes continued to be assembled in VZV-infected cells and were not retained in the endoplasmic reticulum. In contrast, immunofluorescence and confocal microscopy showed that VZV infection resulted in an accumulation of MHC I molecules which colocalized to the Golgi compartment. Inhibition of late viral gene expression by treatment of infected fibroblasts with phosphonoacetic acid did not influence the modulation of MHC I expression, nor did transfection of cells with plasmids expressing immediate early viral proteins. However, cells transfected with a plasmid carrying the early gene ORF66 did result in a significant downregulation of MHC I expression, suggesting that this gene encodes a protein with an immunomodulatory function. Thus, VZV downregulates MHC I expression by impairing the transport of MHC I molecules from the Golgi compartment to the cell surface; this effect may enable the virus to evade CD8+ T-cell immune recognition during VZV pathogenesis, including the critical phase of T-lymphocyte-associated viremia.

Varicella-zoster virus-infected human sensory neurons are resistant to apoptosis, yet human foreskin fibroblasts are susceptible: evidence for a cell-type-specific apoptotic response.

The induction of apoptosis or programmed cell death in virus-infected cells is an important antiviral defense mechanism of the host, and some herpesviruses have evolved strategies to modulate apoptosis in order to enhance their survival and spread. In this study, we examined the ability of varicella-zoster virus (VZV) to induce apoptosis in primary human dorsal root ganglion neurons and primary human foreskin fibroblasts (HFFs). Three independent methods (annexin V, TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] staining, and electron microscopy) were used to assess apoptosis in these cells on days 1, 2, and 4 postinoculation. By all three methods, apoptosis was readily detected in VZV-infected HFFs. In stark contrast, apoptosis was not detected during productive VZV infection of neurons. The low-passage clinical isolate Schenke and the tissue culture-adapted ROka strain both induced apoptosis in HFFs but not in neurons, suggesting that this cell-type-specific apoptotic phenotype was not VZV strain specific. These data show that the regulation of apoptosis differs markedly between HFFs and neurons during productive VZV infection. Inhibition of apoptosis during infection of neurons may play a significant role in the establishment, maintenance, and reactivation of latent infection by promoting survival of these postmitotic cells.

Multicenter evaluation of PCR methods for detecting CMV DNA in blood donors.

BACKGROUND: CMV DNA screening may be a useful adjunct to serologic tests in distinguishing potentially infectious blood donations from those that are "CMV-safe." However, there is currently no consensus on the optimal assay method for accurate detection of CMV DNA in donors. STUDY DESIGN AND METHODS: A blinded multicenter evaluation of seven CMV PCR assays was performed by five laboratories by using coded sets of analytical controls and donor blood samples. RESULTS: Five assays displayed sufficient sensitivity for donor screening, as judged by consistent detection of a minimum of 25 CMV genome equivalents (geq) in analytical controls constructed to contain from 1 to 100 CMV geq in background DNA from 250,000 cells, while the other two assays displayed inadequate sensitivity. Three sensitive assays, two based on nested PCR directed at the UL93 and UL32 regions of the CMV genome and another test (Monitor Assay, Roche), did not detect CMV DNA in samples from any of 20 pedigreed CMV-seronegative, Western blot-negative (S-/WB-) donors. Two other assays based on nested PCR occasionally detected CMV DNA in S-WB- samples, and one sensitive nested PCR assay directed at UL123 detected CMV DNA in a large proportion (85%) of S-WB- samples. CONCLUSION: Seven CMV PCR assays currently used for research and/or diagnostic applications displayed marked variations in sensitivity, specificity, and reproducibility when applied to coded analytical and clinical control samples containing cellular DNA from the equivalent of 250,000 WBCs. These results will be useful in the selection of assays with performance characteristics appropriate to donor screening objectives. They may also help explain discrepant findings from previous studies that used PCR to determine CMV DNA prevalence in seronegative and seropositive blood donors.