Combination immunotherapy with anti-PD-L1 antibody and depletion of regulatory T cells during acute viral infections results in improved virus control but lethal immunopathology

Combination immunotherapy (CIT) is currently applied as a treatment for different cancers and is proposed as a cure strategy for chronic viral infections. Whether such therapies are efficient during an acute infection remains elusive. To address this, inhibitory receptors were blocked and regulatory T cells depleted in acutely Friend retrovirus-infected mice. CIT resulted in a dramatic expansion of cytotoxic CD4+ and CD8+ T cells and a subsequent reduction in viral loads. Despite limited viral replication, mice developed fatal immunopathology after CIT. The pathology was most severe in the gastrointestinal tract and was mediated by granzyme B producing CD4+ and CD8+ T cells. A similar post-CIT pathology during acute Influenza virus infection of mice was observed, which could be prevented by vaccination. Melanoma patients who developed immune-related adverse events under immune checkpoint CIT also presented with expanded granzyme-expressing CD4+ and CD8+ T cell populations. Our data suggest that acute infections may induce immunopathology in patients treated with CIT, and that effective measures for infection prevention should be applied

A Mass Spectrometry-Based Profiling of Interactomes of Viral DDB1- and Cullin Ubiquitin Ligase-Binding Proteins Reveals NF-κB Inhibitory Activity of the HIV-2-Encoded Vpx

Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors

Solution structure of a DNA double helix with consecutive metal-mediated base pairs

Metal-mediated base pairs represent a powerful tool for the site-specific functionlization of nucleic acids with metal ions. The development of applications of the metal-modified nucleic acids will depend on the availability of structural information on these double helices. We present here the NMR solution structure of a self-complementary DNA oligonucleotide with three consecutive imidazole nucleotides in its centre. In the absence of transition-metal ions, a hairpin structure is adopted with the artifical nucleotides forming the loop. In the presence of Ag(I) ions, a duplex comprising three imidazole-Ag+-imidazole base pairs is formed. Direct proof for the formation of metal-mediated base pairs was obtained from (1)J(N-15,Ag-107/109) couplings upon incorporation of N-15-labelled imidazole. The duplex adopts a B-type conformation with only minor deviations in the region of the artifical bases. This work represents the first structural characterization of a metal-modified nucleic acid with a continuous stretch of metal-mediated base pairs

Broad and potent antiviral activity of the NAE inhibitor MLN4924

AbstractIn terms of infected human individuals, herpesviruses range among the most successful virus families. Subclinical herpesviral infections in healthy individuals contrast with life-threatening syndromes under immunocompromising and immunoimmature conditions. Based on our finding that cytomegaloviruses interact with Cullin Roc ubiquitin ligases (CRLs) in the context of interferon antagonism, we systematically assessed viral dependency on CRLs by utilizing the drug MLN4924. CRL activity is regulated through the conjugation of Cullins with the ubiquitin-like molecule Nedd8. By inhibiting the Nedd8-activating Enzyme (NAE), MLN4924 interferes with Nedd8 conjugation and CRL activity. MLN4924 exhibited pronounced antiviral activity against mouse and human cytomegalovirus, herpes simplex virus (HSV)- 1 (including multi-drug resistant clinical isolates), HSV-2, adeno and influenza viruses. Human cytomegalovirus genome amplification was blocked at nanomolar MLN4924 concentrations. Global proteome analyses revealed that MLN4924 blocks cytomegaloviral replication despite increased IE1 amounts. Expression of dominant negative Cullins assigned this IE regulation to defined Cullin molecules and phenocopied the antiviral effect of MLN4924.</jats:p

Data_Sheet_2.PDF

Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors.</p

Data_Sheet_1_A Mass Spectrometry-Based Profiling of Interactomes of Viral DDB1- and Cullin Ubiquitin Ligase-Binding Proteins Reveals NF-κB Inhibitory Activity of the HIV-2-Encoded Vpx.PDF

Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors.</p