Quantitative proteomics defines mechanisms of antiviral defence and cell death during modified vaccinia Ankara infection.

Modified vaccinia Ankara (MVA) virus does not replicate in human cells and is the vaccine deployed to curb the current outbreak of mpox. Here, we conduct a multiplexed proteomic analysis to quantify >9000 cellular and ~80% of viral proteins throughout MVA infection of human fibroblasts and macrophages. >690 human proteins are down-regulated >2-fold by MVA, revealing a substantial remodelling of the host proteome. >25% of these MVA targets are not shared with replication-competent vaccinia. Viral intermediate/late gene expression is necessary for MVA antagonism of innate immunity, and suppression of interferon effectors such as ISG20 potentiates virus gene expression. Proteomic changes specific to infection of macrophages indicate modulation of the inflammatory response, including inflammasome activation. Our approach thus provides a global view of the impact of MVA on the human proteome and identifies mechanisms that may underpin its abortive infection. These discoveries will prove vital to design future generations of vaccines.

Inhibition of cellular RNA methyltransferase abrogates influenza virus capping and replication.

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Brain immune cells undergo cGAS/STING-dependent apoptosis during herpes simplex virus type 1 infection to limit type I IFN production.

Protection of the brain from viral infections involves the type I IFN (IFN-I) system, defects in which render humans susceptible to herpes simplex encephalitis (HSE). However, excessive cerebral IFN-I levels lead to pathologies, suggesting the need for tight regulation of responses. Based on data from mouse models, human HSE cases, and primary cell culture systems, we showed that microglia and other immune cells undergo apoptosis in the HSV-1-infected brain through a mechanism dependent on the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, but independent of IFN-I. HSV-1 infection of microglia induced cGAS-dependent apoptosis at high viral doses, whereas lower viral doses led to IFN-I responses. Importantly, inhibition of caspase activity prevented microglial cell death and augmented IFN-I responses. Accordingly, HSV-1-infected organotypic brain slices or mice treated with a caspase inhibitor exhibited lower viral load and an improved infection outcome. Collectively, we identify an activation-induced apoptosis program in brain immune cells that downmodulates local immune responses.

Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells.

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.

Lifestyle Intervention in Danish Obese Pregnant Women With Early Gestational Diabetes Mellitus According to WHO 2013 Criteria Does Not Change Pregnancy Outcomes: Results From the LiP (Lifestyle in Pregnancy) Study.

OBJECTIVE: To study effects of lifestyle intervention on metabolic and clinical outcomes in obese women fulfilling the World Health Organization (WHO) 2013 diagnostic criteria for gestational diabetes mellitus (GDM) in early gestation. RESEARCH DESIGN AND METHODS: Secondary analysis of data from the Lifestyle in Pregnancy (LiP) study, a lifestyle randomized controlled trial in 304 pregnant women with BMI ≥30 kg/m2. Early GDM (week 12-15) was diagnosed according to modified WHO 2013 GDM criteria: fasting venous plasma glucose ≥5.1 mmol/L and/or 2-h capillary blood glucose (CBG) ≥8.5 mmol/L (75-g oral glucose tolerance test [OGTT]). Women with treated GDM fulfilling local Danish GDM criteria (2-h CBG ≥9.0 mmol/L) (n = 16) and women with normal OGTT (n = 198) were excluded. RESULTS: Of 90 women with early GDM, 36 received lifestyle intervention and 54 standard care. All were Caucasian, and median age was 29 years (interquartile range 27-33) and BMI 34.5 kg/m2 (32.3-38.1). All baseline characteristics were similar in the lifestyle intervention and standard care groups. At gestational week 28-30, the women in the lifestyle intervention group had significantly higher fasting total cholesterol and fasting LDL. All other metabolic parameters including measurements of glucose, insulin, and HOMA of insulin resistance were similar. There were more planned cesarean sections in the lifestyle intervention group (22.2 vs. 5.6%), but all other obstetric outcomes were similar. CONCLUSIONS: Lifestyle intervention in obese women fulfilling WHO 2013 GDM criteria in early pregnancy was not effective in improving obstetric or metabolic outcomes. Future studies should focus on interventions starting prepregnancy.

Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1.

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.

cGAS is activated by DNA in a length-dependent manner.

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP-AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length-dependent manner. This is observed over a wide length-span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length-dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.

Viral evasion of DNA-stimulated innate immune responses.

Cellular sensing of virus-derived nucleic acids is essential for early defenses against virus infections. In recent years, the discovery of DNA sensing proteins, including cyclic GMP-AMP synthase (cGAS) and gamma-interferon-inducible protein (IFI16), has led to understanding of how cells evoke strong innate immune responses against incoming pathogens carrying DNA genomes. The signaling stimulated by DNA sensors depends on the adaptor protein STING (stimulator of interferon genes), to enable expression of antiviral proteins, including type I interferon. To facilitate efficient infections, viruses have evolved a wide range of evasion strategies, targeting host DNA sensors, adaptor proteins and transcription factors. In this review, the current literature on virus-induced activation of the STING pathway is presented and we discuss recently identified viral evasion mechanisms targeting different steps in this antiviral pathway.

HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression.

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING.

The innate immune system senses infection by detecting either evolutionarily conserved molecules essential for the survival of microbes or the abnormal location of molecules. Here we demonstrate the existence of a previously unknown innate detection mechanism induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon response with expression of interferon-stimulated genes, in vivo recruitment of leukocytes and potentiation of signaling via Toll-like receptor 7 (TLR7) and TLR9. The fusion-dependent response was dependent on the stimulator of interferon genes STING but was independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant-cell formation.