Angeborene Immunität gegen und Virale Evasionsmechanismen von HIV-1 und SARS-CoV-2

The innate immune system is the first line of defence against invading pathogens. Pattern recognition receptors (PRRs) sense conserved pathogen-associated molecular patterns (PAMPs) and initiate a signal transduction cascade that culminates in the secretion of type I interferons (IFNs), pro-inflammatory cytokines and amplification of innate immunity through autocrine and paracrine signalling. Type I IFNs are universal, antiviral cytokines that bind to the IFN-α/β-receptor (IFNAR) and induce the JAK/STAT-dependent expression of hundreds of antiviral molecules, known as IFN-stimulated genes (ISGs). Aberrantly located, cytosolic DNA is a potent activator of type I IFN responses upon detection by the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS). Upon DNA sensing, cGAS catalyses the formation of the second messenger cyclic GMP-AMP (cGAMP) that subsequently induces a STING/TBK1/IRF3-driven signalling cascade and the production of type I IFNs. Early initiation of intrinsic immunity is crucial for viral restriction, elimination of infected cells and orchestration of adaptive immunity to sufficiently control the viral infection and dampen disease progression in vivo. Consequently, multiple viruses have evolved mechanisms to counteract restrictive type I IFN immunity to facilitate continuous viral replication, e.g. through expression of antagonistic viral proteins that modulate PRR-induced signalling or the development of sophisticated replication strategies to minimize viral PAMP exposure to cellular PRRs. This dissertation is based on my three recent publications addressing key questions in the field of innate sensing and viral evasion of HIV-1 and SARS-CoV-2 infections. The first study investigated the structural and functional consequences of the most frequent naturally occurring variant rs610913 in the cGAS-encoding gene in the context of DNA sensing upon viral infections (1). The second study carefully re-evaluates the contribution of cGAS-mediated sensing of reverse transcription products to the induction of intrinsic immunity in HIV-1-infected CD4+ T-cells, the major HIV-1 target cell in vivo (2). In the third study, the susceptibility to infection and ability to mount cell-intrinsic immunity in peripheral blood mononuclear cells upon ex vivo SARS-CoV-2 exposure were investigated and delineated to individual cell types using, among other methods, single cell RNA-sequencing (3).Das angeborene Immunsystem ist die erste Abwehrlinie gegen eindringende Pathogene. Zelluläre Mustererkennungsrezeptoren (engl. PRRs) detektieren konservierte Pathogen assoziierte molekulare Muster (engl. PAMPs) und initiieren eine Signaltransduktionskaskade, die in der Sekretion von Typ I Interferonen (IFN) und pro inflammatorischen Zytokinen mündet, welche durch parakrine und autokrineMechanismen die Immunantwort amplifizieren. Typ I IFN sind universelle, antivirale Zytokine, die nach Bindung an den IFN-α/β-Rezeptor die JAK/STAT-abhängige Expression hunderter antiviraler Moleküle, der so genannten IFN-stimulierten Gene, auslöst. Irrtümlich im zellulären Zytosol lokalisierte DNA ist ein besonders potenter Auslöser von Typ I IFN-vermittelter Immunität. Das Enzym zyklische GMP-AMP Synthase (engl. cGAS) katalysiert nach Bindung an zytosolischer DNA die Synthese des sekundären Botenstoffes zyklisches GMP-AMP (cGAMP), welches eine STING/TBK1/IRF3-abhängige Signalkaskade zur Produktion von Typ I IFN auslöst. Die frühe Initiierung einer solchen angeborenen Immunantwort ist entscheidend für die Restriktion viraler Replikation, die Eliminierung infizierter Zellen und die Einleitung der adaptiven Immunantwort zur effizienten Kontrolle der Infektion und der Abmilderung des Krankheitsverlaufs in vivo. Folglich haben Viren Evasionsmechanismen entwickelt, um der Typ I IFN-vermittelten Restriktion zu entgehen und die virale Replikation zu fördern, zum Beispiel durch die Expression antagonistischer viraler Proteine, die PRR-ausgelöste Signalketten zum Erliegen bringen oder durch die Entwicklung anspruchsvoller Replikationsstrategien, die die Exposition viraler PAMPs gegenüber den zellulären PRRs auf ein Minimum reduziert. Diese Dissertation basiert auf den drei kürzlich unter meiner maßgeblichen Mitarbeit veröffentlichten Publikationen, die jeweils Schlüsselfragen der angeborenen Immunität und viraler Evasion im Kontext der HIV-1- und SARS-CoV-2-Infektion adressieren. Die erste Studie untersucht die strukturellen und funktionellen Konsequenzen der am häufigsten natürlich vorkommenden cGAS-Variante, rs610913, in Bezug auf deren Fähigkeit DNA-vermittelte Immunantworten im Kontext viraler Infektionen auszulösen (1). Die zweite Studie evaluiert den Beitrag der cGAS-vermittelten Detektion proviraler DNA zur intrinsischen Immunantwort in HIV-1-infizierten CD4+ T-Zellen, den Hauptzielzellen von HIV-1 in vivo (2). In der dritten Studie wird die Infektionsempfänglichkeit und initiierte angeborene Immunantwort in peripheren mononukleären Zellen des Blutes nach ex vivo Inokulation mit SARS-CoV-2 unter Anderem mittels Einzelzellsequenzierung analysiert (3)

Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients’ cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha

Absence of cGAS-mediated type I IFN responses in HIV-1-infected T cells

The DNA sensor cGAS catalyzes the production of the cyclic dinucleotide cGAMP, resulting in type I interferon responses. We addressed the functionality of cGAS-mediated DNA sensing in human and murine T cells. Activated primary CD4(+) T cells expressed cGAS and responded to plasmid DNA by upregulation of ISGs and release of bioactive interferon. In mouse T cells, cGAS KO ablated sensing of plasmid DNA, and TREX1 KO enabled cells to sense short immunostimulatory DNA. Expression of IFIT1 and MX2 was downregulated and upregulated in cGAS KO and TREX1 KO T cell lines, respectively, compared to parental cells. Despite their intact cGAS sensing pathway, human CD4(+) T cells failed to mount a reverse transcriptase (RT) inhibitor-sensitive immune response following HIV-1 infection. In contrast, infection of human T cells with HSV-1 that is functionally deficient for the cGAS antagonist pUL41 (HSV-1 Delta UL41N) resulted in a cGAS-dependent type I interferon response. In accordance with our results in primary CD4(+) T cells, plasmid challenge or HSV-1 Delta UL41N inoculation of T cell lines provoked an entirely cGAS-dependent type I interferon response, including IRF3 phosphorylation and expression of ISGs. In contrast, no RT-dependent interferon response was detected following transduction of T cell lines with VSV-G-pseudotyped lentiviral or gammaretroviral particles. Together, T cells are capable to raise a cGAS-dependent cell-intrinsic response to both plasmid DNA challenge or inoculation with HSV-1 Delta UL41N. However, HIV-1 infection does not appear to trigger cGAS-mediated sensing of viral DNA in T cells, possibly by revealing viral DNA of insufficient quantity, length, and/or accessibility to cGAS

Synthesis and evaluation of novel a-amino cyclic boronates as inhibitors of HCV NS3 protease

We have designed and synthesized a novel series of alpha-amino cyclic boronates and incorporated them successfully in several acyclic templates at the P1 position. These compounds are inhibitors of the HCV NS3 serine protease, and structural studies show that they inhibit the NS3 protease by trapping the Ser-139 hydroxyl group in the active site. Synthetic methodologies and SARs of this series of compounds are described

Novel macrocyclic HCV NS3 protease inhibitors derived from a-amino cyclic boronates

A novel series of P2-P4 macrocyclic HCV NS3/4A protease inhibitors with a-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around a-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern. Furthermore, X-ray structure of the co-crystal of inhibitor 9a and NS3 protease revealed that Ser-139 in the enzyme active site traps boron in the warhead region of 9a, thus establishing its mode of action

Delayed cytopathic onset of VOC Alpha SARS-CoV-2 infection.

Vero E6 cells were infected with B.1, VOC Alpha/v1, and VOC Alpha/v2 (MOI 0.001). Onset of CPE was monitored by live cell imaging until 70 hours postinfection. CPE, cytopathogenic effect; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VOC, variant of concern. (MP4)</p