Activation of interferon regulatory factor (IRF)-3 and/or IRF-7 drives the expression of antiviral genes and the production of a/b IFN, a hallmark of antiviral responses triggered by Toll-like receptors (TLR). Here we describe a novel antiviral signaling pathway operating in myeloid (m) dendritic cells (DC) and macrophages that does not require IRF-3 and/or IRF-7 but is driven by IRF-1. IRF-1 together with myeloid differentiation factor 88 (MyD88) or IL-1 receptor-associated kinase (IRAK)-1 triggered IFN-b promoter activation. IRF-1 physically interacted with MyD88 and activation of mDC via TLR-9 induced IRF-1-dependent IFN-b production paralleled by rapid transcriptional activation of IFN-stimulated genes. The NF-jB-dependent production of pro-inflammatory cytokines, however, was not influenced by IRF-1. TLR-9 signaling through this pathway conferred cellular antiviral resistance while IRF-1-deficient mice displayed enhanced susceptibility to viral infection. These results demonstrate that TLR-9 activation of mDC and macrophages contributes to antiviral immunity via IRF-1. See accompanying commentary: http://dx

The human gammaherpesviruses Kaposi’s sarcoma-associated herpesvirus and EBV cause important infections. As pathogenetic studies of the human infections are restricted, murine gammaherpesvirus 68 serves as a model to study gammaherpesvirus pathogenesis. TLRs are a conserved family of receptors detecting microbial molecular patterns. Among the TLRs, TLR9 recognizes unmethylated CpG DNA motifs present in bacterial and viral DNA. The aim of this study was to assess the role of TLR9 in gammaherpesvirus pathogenesis. Upon stimulation with murine gammaherpesvirus 68, Flt3L-cultured bone marrow cells (dendritic cells) from TLR9−/− mice secreted reduced levels of IL-12, IFN-α, and IL-6, when compared with dendritic cells from wild-type mice. Intranasal infection of TLR9−/− and wild-type mice did not reveal any differences during lytic and latent infection. In contrast, when infected i.p., TLR9−/− mice showed markedly higher viral loads both during lytic and latent infection. Thus, we show for the first time that TLR9 is involved in gammaherpesvirus pathogenesis and contributes to organ-specific immunity.

Background: Diseases caused by gammaherpesviruses continue to be a challenge for human health and antiviral treatment. Most of the commonly used antiviral drugs are directed against viral gene products. However, the emergence of drug-resistant mutations ma limit the effectiveness of these drugs. Since viruses require a host cell to propagate, the search for host cell targets is an interestin alternative. Methods: In this study, we infected three different cell types (fibroblasts, endothelial precursor cells and macrophages with a murine gammaherpesvirus and analysed the host cell response for changes either common to all or unique to a particular cell type using oligonucleotide microarrays. Results: The analysis revealed a number of genes whose transcription was significantly up- or down-regulated in either one or two of the cell types tested. After infection, only two genes, Lman1 (also known as ERGIC53) an synaptobrevin-like 1 (sybl1) were significantly up-regulated in all three cell types, suggestive for a general role for the virus life cycl independent of the cell type. Both proteins have been implicated in cellular exocytosis and transport of glycoproteins through the secre tory pathway. To test the significance of the observed up-regulation, the functionality of these proteins was modulated, and the effect on virus replication was monitored. Inhibition of either Lman1 or sybl1 resulted in a significant reduction in virus production. Conclusions: This suggests that proteins of the secretory pathway which appear to be rate limiting for virus production may represent new targets for intervention.

Control of gammaherpesvirus infections requires a complex, well orchestrated immune response regulated by positive and negative co-signaling molecules. While the impact of co-stimulatory molecules has been addressed in various studies, the role of co-inhibitory receptors has not been tested. The ITIM-bearing CEACAM1 is an inhibitory receptor expressed by a variety of immune cells, including B, T and NK cells. Using Ceacam1−/− mice, we analyzed the in vivo function of CEACAM1 during acute and latent murine gammaherpesvirus 68 (MHV-68) infection. During acute lytic replication, we observed lower virus titers in the lungs of Ceacam1−/− mice than in WT mice. In contrast, during latency amplification, Ceacam1−/− mice displayed increased splenomegaly and a higher latent viral load in the spleen. Analysis of the immune response revealed increased virus-specific antibody levels in Ceacam1−/− mice, while the magnitude of the T cell-mediated antiviral immune response was reduced. These findings suggest that inhibitory receptors can modulate the efficacy of immune responses against gammaherpesvirus infections.

The aim of the present study was to identify and functionally characterize the equine herpesvirus 1 (EHV-1) UL20 protein (UL20p). Using a specific antiserum, UL20p was shown to be associated with membranes of infected cells, as well as with envelopes of purified virions. By Western blot analysis, UL20p was detected in two main forms exhibiting M r s of 25,000 and 75,000. Both moieties did not enter the separating gel after heating of protein samples to 99°C. The slower-migrating form of UL20p contains N-linked carbohydrates, and its presence is dependent of that of other viral proteins. Infection of cells that either constitutively express UL20p or a gK-green fluorescent protein (GFP) fusion protein with various EHV-1 deletion mutants revealed a relatively stable hetero-oligomer containing gK and UL20p with an apparent M r of 75,000. As demonstrated by confocal microscopy, UL20p distribution in Rk13 cells changed from a diffuse granular or netlike appearance to a pattern confined to the Golgi network when gK was coexpressed. Analysis of a UL20 deletion mutant of EHV-1 strain RacL11 indicated an involvement of UL20p in cell-to-cell spread, as well as in very late events in virus egress. Based on these and electron microscopic studies we suggest that the EHV-1 UL20 protein might be necessary to avoid fusion of mature virions with membranes of their transport vesicles. Equid herpesvirus 1 (equine herpesvirus 1 [EHV-1]) is one of the most important viral pathogens of horses and classifies as a Varicellovirus within the subfamily Alphaherpesvirinae (21).Alphaherpesviruses express relatively few proteins with the potential to span membranes several times. Accordingly, only four such open reading frames (ORFs) have been identified in the EHV-1 sequence, and they are homologous to the herpes simplex virus type 1 (HSV-1) ORFs UL10, UL20, UL43, and UL53 (31). Of these, the EHV-1 UL10 product glycoprotein M (gM), has been studied most intensively, and it has been shown that gM is involved in virus egress, more precisely in secondary envelopment, and plays a role in cell-to-cell spread (23, 28). The protein encoded by UL53, gK, is less dispensable for virus replication in Rk13 cells than gM. However, like gM, gK influences a late step in virus egress, direct cell-to-cell spread, and the efficiency of virus penetration (20). EHV-1 gK and gM share additional characteristics such as N glycosylation, earlylate expression kinetics, and the fact that they both depend on a viral complex partner for complete processing (20,25). In the present study we sought to characterize the UL20 product to further investigate the specific roles of multiply hydrophobic proteins in EHV-1 replication and to address the question whether the special structural features might be related to common functional properties.In contrast to the EHV-1 UL20 protein, which has not been studied before, the respective homologs of HSV-1 and pseudorabies virus (PRV) are well characterized. The structure of the PRV UL20p is still unknown, since no specific antibodie...