The chaperone protein Unc-93 homolog B1 (UNC93B1) regulates internalization, trafficking, and stabilization of nucleic acid-sensing Toll-like receptors (TLR) in peripheral immune cells. We sought to determine UNC93B1 expression and its functional relevance in inflammatory and injurious processes in the central nervous system (CNS). We found that UNC93B1 is expressed in various CNS cells including microglia, astrocytes, oligodendrocytes, and neurons, as assessed by PCR, immunocyto-/histochemistry, and flow cytometry. UNC93B1 expression in the murine brain increased during development. Exposure to the microRNA let-7b, a recently discovered endogenous TLR7 activator, but also to TLR3 and TLR4 agonists, led to increased UNC93B1 expression in microglia and neurons. Microglial activation by extracellular let-7b required functional UNC93B1, as assessed by TNF ELISA. Neuronal injury induced by extracellular let-7b was dependent on UNC93B1, as UNC93B1-deficient neurons were unaffected by the microRNA's neurotoxicity in vitro. Intrathecal application of let-7b triggered neurodegeneration in wild-type mice, whereas mice deficient for UNC93B1 were protected against injurious effects on neurons and axons. In summary, our data demonstrate broad UNC93B1 expression in the murine brain and establish this chaperone as a modulator of neuroinflammation and neuronal injury triggered by extracellular microRNA and subsequent induction of TLR signaling.

Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, ZAP-S and ZAP-L, is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP results in reduced viral mRNA and protein levels and decelerates the progression of HCMV infection. Metabolic RNA labeling combined with high-throughput sequencing (SLAM-seq) revealed that most of the gene expression changes late in infection result from the general attenuation of HCMV. Furthermore, at early stages of infection, ZAP restricts HCMV by destabilizing a distinct subset of viral mRNAs, particularly those from the previously uncharacterized UL4-UL6 HCMV gene locus. Through enhanced cross-linking immunoprecipitation and sequencing analysis (eCLIP-seq), we identified the transcripts expressed from this HCMV locus as the direct targets of ZAP. Moreover, our data show that ZAP preferentially recognizes not only CG, but also other cytosine-rich sequences, thereby expanding its target specificity. In summary, this report is the first to reveal direct targets of ZAP during HCMV infection, which strongly indicates that transcripts from the UL4-UL6 locus may play an important role for HCMV replication.IMPORTANCE Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. There is no licensed HCMV vaccine in sight to protect those most at risk, particularly immunocompromised individuals or pregnant women who might otherwise transmit the virus to the fetus. Thus, the identification of novel intervention strategies is urgently required. In this study, we show that ZAP decelerates the viral gene expression cascade, presumably by selectively handpicking a distinct set of viral transcripts for degradation. Our study illustrates the potent role of ZAP as an HCMV restriction factor and sheds light on a possible role for UL4 and/or UL5 early during infection, paving a new avenue for the exploration of potential targets for novel therapies.

We describe a multifactorial investigation of a SARS-CoV-2 outbreak in a large meat processing complex in Germany. Infection event timing, spatial, climate and ventilation conditions in the processing plant, sharing of living quarters and transport, and viral genome sequences were analyzed. Our results suggest that a single index case transmitted SARS-CoV-2 to co-workers over distances of more than 8 meters, within a confined work area in which air is constantly recirculated and cooled. Viral genome sequencing shows that all cases share a set of mutations representing a novel sub-branch in the SARS-CoV-2 C20 clade. We identified the same set of mutations in samples collected in the time period between this initial infection cluster and a subsequent outbreak within the same factory, with the largest number of confirmed SARS-CoV-2 cases in a German meat processing facility reported so far. Our results indicate climate conditions, fresh air exchange rates, and airflow as factors that can promote efficient spread of SARS-CoV-2 via long distances and provide insights into possible requirements for pandemic mitigation strategies in industrial workplace settings.

The host immune system is engaged in a constant battle with microorganisms, with the immediate detection of pathogenic invasion and subsequent signalling acting as crucial deterrents against the establishment of a successful infection. For this purpose, cells are equipped with a variety of sensors called pattern recognition receptors (PRR), which rapidly detect intruders leading to the expression of antiviral type I interferons (IFN). Type I IFN are crucial cytokines which exert their biological effects through the induction of hundreds of IFN-stimulated genes (ISGs). The expression profile of these ISGs varies depending on the virus. For a small subset of ISGs, their anti- or even proviral effects have been revealed, however, the vast majority are uncharacterised. The spotlight is now on herpesviruses, with their large coding capacity and long co-evolution with their hosts, as a key to understanding the impact of ISGs during viral infection. Studies are emerging which have identified multiple herpesviral antagonists specifically targeting ISGs, hinting at the significant role these proteins must play in host defence against viral infection, with the promise of more to come. In this review, we will discuss the current knowledge of the complex interplay between ISGs and human herpesviruses: the antiviral role of selected ISGs during herpesviral infections, how herpesviruses antagonise these ISGs and, in some cases, even exploit them to benefit viral infection.

Murine gammaherpesvirus 68 (MHV68) is an amenable small animal model for study of the human pathogens Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal TLR escort protein UNC93B, endosomal TLR7, 9, and 13, and cell surface TLR2 in MHV68 detection. We found that the interferon α (IFNα) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9-/- cells compared to wildtype (WT), but not completely lost. Tlr7-/- pDC responded similarly to WT. However, we found that in Unc93b-/- pDC, as well as in Tlr7/Tlr9-/- double knockout pDC, the IFNα response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFNα response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7/Tlr9-/- mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b-/-, Tlr7/Tlr9-/-, Tlr7-/-, and Tlr9-/- splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7/Tlr9-/- splenocytes, compared to WT. Our data show that UNC93B- dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few oncogenic human viruses known to date. Its large genome encodes more than 85 proteins and includes both unique viral proteins as well as proteins conserved amongst herpesviruses. KSHV ORF20 is a member of the herpesviral core UL24 family, but the function of ORF20 and its role in the viral life cycle is not well understood. ORF20 encodes three largely uncharacterized isoforms, which we found were localized predominantly in the nuclei and nucleoli. Quantitative affinity purification coupled to mass spectrometry (q-AP-MS) identified numerous specific interacting partners of ORF20, including ribosomal proteins and the interferon-stimulated gene product (ISG) oligoadenylate synthetase-like protein (OASL). Both endogenous and transiently transfected OASL co-immunoprecipitated with ORF20, and this interaction was conserved among all ORF20 isoforms and multiple ORF20 homologs of the UL24 family in other herpesviruses. Characterization of OASL interacting partners by q-AP-MS identified a very similar interactome to that of ORF20. Both ORF20 and OASL copurified with 40S and 60S ribosomal subunits, and when they were co-expressed, they associated with polysomes. Although ORF20 did not have a global effect on translation, ORF20 enhanced RIG-I induced expression of endogenous OASL in an IRF3-dependent but IFNAR-independent manner. OASL has been characterized as an ISG with antiviral activity against some viruses, but its role for gammaherpesviruses was unknown. We show that OASL and ORF20 mRNA expression were induced early after reactivation of latently infected HuARLT-rKSHV.219 cells. Intriguingly, we found that OASL enhanced infection of KSHV. During infection with a KSHV ORF20stop mutant, however, OASL-dependent enhancement of infectivity was lost. Our data have characterized the interaction of ORF20 with OASL and suggest ORF20 usurps the function of OASL to benefit KSHV infection.

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tail-anchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction.

The coordinated action of a variety of virulence factors allows Salmonella enterica to invade epithelial cells and penetrate the mucosal barrier. The influence of the age-dependent maturation of the mucosal barrier for microbial pathogenesis has not been investigated. Here, we analyzed Salmonella infection of neonate mice after oral administration. In contrast to the situation in adult animals, we observed spontaneous colonization, massive invasion of enteroabsorptive cells, intraepithelial proliferation and the formation of large intraepithelial microcolonies. Mucosal translocation was dependent on enterocyte invasion in neonates in the absence of microfold (M) cells. It further resulted in potent innate immune stimulation in the absence of pronounced neutrophil-dominated pathology. Our results identify factors of age-dependent host susceptibility and provide important insight in the early steps of Salmonella infection in vivo. We also present a new small animal model amenable to genetic manipulation of the host for the analysis of the Salmonella enterocyte interaction in vivo.

The production of cytokines is a crucial element of the host response to viral and bacterial infections. To follow these events in vivo, transgenic mice have become a valuable tool to study cytokine production through induction of reporter genes. We describe here the generation and immortalization of cells derived from transgenic reporter mice for development of a high-throughput assay system for virus- or bacteria-induced cytokine induction. As an example we describe mouse cytomegalovirus (MCMV) infection of immortalized fibroblasts derived from mice expressing the firefly luciferase reporter downstream of the IFN-β promoter. Common methods to determine IFN-β production, including ELISA, quantitative real-time PCR (qPCR), and transient reporter assays using plasmid-based reporter constructs, have disadvantages and limitations. Transient transfections influence type I IFN responses in most cell types, and IFN-β ELISA as well as qPCR are both laborious and expensive. The method presented here is highly sensitive as well as cost-effective, and allows monitoring of a robust and dose-dependent induction of IFN-β upon virus infection in cell lysates as well as living cells.

Toll-like receptor-9 (TLR9) is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the pathogenic secretion of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Here, we found that in response to DNA-containing immune complexes (DNA-IC), but not to soluble ligands, IFN-α production depended upon the convergence of the phagocytic and autophagic pathways, a process called microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP). LAP was required for TLR9 trafficking into a specialized interferon signaling compartment by a mechanism that involved autophagy-related proteins, but not the conventional autophagic preinitiation complex, or adaptor protein-3 (AP-3). Our findings unveil a new role for nonconventional autophagy in inflammation and provide one mechanism by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict the apportionment of pathogenic DNA and TLR9 to the interferon signaling compartment.