cGAMP Quantification in Virus-Infected Human Monocyte-Derived Cells by HPLC-Coupled Tandem Mass Spectrometry.

Upon virus infection, cells of the innate immune system such as dendritic cells and macrophages can mount type I interferon (IFN-I) responses that restrict viral dissemination. To inform host cells of virus infection, detection of cytosolic DNA is one important mechanism. Inappropriate sensing of endogenous DNA and subsequent induction of IFN-I responses can also cause autoimmunity, highlighting the need to tightly regulate DNA sensing. The cyclic GMP-AMP synthase (cGAS) was recently identified to be the major sensor of cytosolic DNA that triggers IFN-I expression. Upon DNA binding, cGAS synthesizes the second messenger cyclic guanosine-adenosine monophosphate (cGAMP) that induces IFN-I expression by the activation of the stimulator of interferon genes (STING). Notably, cGAMP does not only act in infected cells, but can also be relocated to noninfected bystander cells to there trigger IFN-I expression. Thus, direct quantification of cGAMP in cells of the innate immune system is an important approach to study where, when, and how DNA is sensed and IFN-I responses are induced. Here, we describe a method that allows specific quantification of cGAMP from extracts of virus-infected human myeloid cells by HPLC-coupled tandem mass spectrometry.

A highly conserved sequence of the viral TAP inhibitor ICP47 is required for freezing of the peptide transport cycle.

The transporter associated with antigen processing (TAP) translocates antigenic peptides into the endoplasmic reticulum (ER) lumen for loading onto MHC class I molecules. This is a key step in the control of viral infections through CD8+ T-cells. The herpes simplex virus type-1 encodes an 88 amino acid long species-specific TAP inhibitor, ICP47, that functions as a high affinity competitor for the peptide binding site on TAP. It has previously been suggested that the inhibitory function of ICP47 resides within the N-terminal region (residues 1-35). Here we show that mutation of the highly conserved 50PLL52 motif within the central region of ICP47 attenuates its inhibitory capacity. Taking advantage of the human cytomegalovirus-encoded TAP inhibitor US6 as a luminal sensor for conformational changes of TAP, we demonstrated that the 50PLL52 motif is essential for freezing of the TAP conformation. Moreover, hierarchical functional interaction sites on TAP dependent on 50PLL52 could be defined using a comprehensive set of human-rat TAP chimeras. This data broadens our understanding of the molecular mechanism underpinning TAP inhibition by ICP47, to include the 50PLL52 sequence as a stabilizer that tethers the TAP-ICP47 complex in an inward-facing conformation.

A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry.

The large scale of the Ebola virus disease (EVD) outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the responsible Ebola virus (EBOV) strain differed from those of other ebolaviruses. We previously reported that the glycoprotein (GP) of the virus circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two nonhuman primate (NHP)-derived cell lines relative to the GP of EBOV1976. Here, we investigated the molecular determinants underlying the differential entry efficiency. We found that EBOV2014-GP-driven entry into diverse NHP-derived cell lines, as well as human monocyte-derived macrophages and dendritic cells, was reduced compared to EBOV1976-GP, although entry into most human- and all bat-derived cell lines tested was comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976, suggesting that reduced cell entry translated into reduced viral spread. Mutagenic analysis of EBOV2014-GP and EBOV1976-GP revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell line-independent manner and did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast, polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry phenotype. These results suggest that position 544 is an important determinant of EBOV infectivity for both NHP and certain human target cells.IMPORTANCE The Ebola virus disease outbreak in West Africa in 2013 entailed more than 10,000 deaths. The scale of the outbreak and its dramatic impact on human health raised the question whether the responsible virus was particularly adept at infecting human cells. Our study shows that an amino acid exchange, A82V, that was acquired during the epidemic and that was not observed in previously circulating viruses, increases viral entry into diverse target cells. In contrast, the epidemic virus showed a reduced ability to enter cells of nonhuman primates compared to the virus circulating in 1976, and a single amino acid exchange in the internal fusion loop of the viral glycoprotein was found to account for this phenotype.

Concomitant TLR/RLH signaling of radioresistant and radiosensitive cells is essential for protection against vesicular stomatitis virus infection.

Several studies indicated that TLR as well as retinoic acid-inducible gene I-like helicase (RLH) signaling contribute to vesicular stomatitis virus (VSV)-mediated triggering of type I IFN (IFN-I) responses. Nevertheless, TLR-deficient MyD88(-/-)Trif(-/-) mice and RLH-deficient caspase activation and recruitment domain adaptor inducing IFN-ß (Cardif)(-/-) mice showed only marginally enhanced susceptibility to lethal VSV i.v. infection. Therefore, we addressed whether concomitant TLR and RLH signaling, or some other additional mechanism, played a role. To this end, we generated MyD88(-/-)Trif(-/-)Cardif(-/-) (MyTrCa(-/-)) mice that succumbed to low-dose i.v. VSV infection with similar kinetics as IFN-I receptor-deficient mice. Three independent approaches (i.e., analysis of IFN-α/ß serum levels, experiments with IFN-ß reporter mice, and investigation of local IFN-stimulated gene induction) revealed that MyTrCa(-/-) mice did not mount IFN-I responses following VSV infection. Of note, treatment with rIFN-α protected the animals, qualifying MyTrCa(-/-) mice as a model to study the contribution of different immune cell subsets to the production of antiviral IFN-I. Upon adoptive transfer of wild-type plasmacytoid dendritic cells and subsequent VSV infection, MyTrCa(-/-) mice displayed significantly reduced viral loads in peripheral organs and showed prolonged survival. On the contrary, adoptive transfer of wild-type myeloid dendritic cells did not have such effects. Analysis of bone marrow chimeric mice revealed that TLR and RLH signaling of radioresistant and radiosensitive cells was required for efficient protection. Thus, upon VSV infection, plasmacytoid dendritic cell-derived IFN-I primarily protects peripheral organs, whereas concomitant TLR and RLH signaling of radioresistant stroma cells as well as of radiosensitive immune cells is needed to effectively protect against lethal disease.