Physical Requirements and Functional Consequences of Complex Formation between the Cytomegalovirus IE1 Protein and Human STAT2

ABSTRACT Our previous work has shown that efficient evasion from type I interferon responses by human cytomegalovirus (hCMV) requires expression of the 72-kDa immediate-early 1 (IE1) protein. It has been suggested that IE1 inhibits interferon signaling through intranuclear sequestration of the signal transducer and activator of transcription 2 (STAT2) protein. Here we show that physical association and subnuclear colocalization of IE1 and STAT2 depend on short acidic and serine/proline-rich low-complexity motifs in the carboxy-terminal region of the 491-amino-acid viral polypeptide. These motifs compose an essential core (amino acids 373 to 420) and an adjacent ancillary site (amino acids 421 to 445) for STAT2 interaction that are predicted to form part of a natively unstructured domain. The presence of presumably “disordered” carboxy-terminal domains enriched in low-complexity motifs is evolutionarily highly conserved across all examined mammalian IE1 orthologs, and the murine cytomegalovirus IE1 protein appears to interact with STAT2 just like the human counterpart. A recombinant hCMV specifically mutated in the IE1 core STAT2 binding site displays hypersensitivity to alpha interferon, delayed early viral protein accumulation, and attenuated growth in fibroblasts. However, replication of this mutant virus is specifically restored by knockdown of STAT2 expression. Interestingly, complex formation with STAT2 proved to be entirely separable from disruption of nuclear domain 10 (ND10), another key activity of IE1. Finally, our results demonstrate that IE1 counteracts the antiviral interferon response and promotes viral replication by at least two distinct mechanisms, one depending on sequestration of STAT2 and the other one likely involving ND10 interaction.

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NSUN2-mediated m5C methylation of IRF3 mRNA negatively regulates type I interferon responses

10.1101/2021.07.09.451748 ◽

2021 ◽

Author(s):

Hongyun Wang ◽

Lu Zhang ◽

Cong Zeng ◽

Jiangpeng Feng ◽

Yu Zhou ◽

...

Keyword(s):

Viral Infection ◽

Type I Interferon ◽

Sendai Virus ◽

Negative Regulator ◽

Interferon Response ◽

Rna Modification ◽

Type I ◽

Mrna Levels ◽

Interferon Responses

5-Methylcytosine (m5C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m5C methyltransferase, can negatively regulate type I interferon responses during viral infection. NSUN2 specifically mediates m5C methylation of IRF3 mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-β production. Knockout or knockdown of NSUN2 could enhance type I interferon responses and downstream ISG expression after viral infection in vitro. And in vivo, the antiviral innate responses is more dramatically enhanced in Nsun2+/− mice than in Nsun2+/+ mice. Four highly m5C methylated cytosines in IRF3 mRNA were identified, and their mutation could enhance the cellular IRF3 mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), Zika virus (ZIKV), or especially SARS-CoV-2 resulted in a reduction in endogenous levels of NSUN2. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of IRF3 mRNA, while endogenous NSUN2 levels decrease after viral infection to boost antiviral responses for the effective elimination of viruses. Our results suggest a paradigm of innate antiviral immune responses ingeniously involving NSUN2-mediated m5C modification.

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Siglec-H-Deficient Mice Show Enhanced Type I IFN Responses, but Do Not Develop Autoimmunity After Influenza or LCMV Infections

Frontiers in Immunology ◽

10.3389/fimmu.2021.698420 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nadine Szumilas ◽

Odilia B. J. Corneth ◽

Christian H. K. Lehmann ◽

Heike Schmitt ◽

Svenia Cunz ◽

...

Keyword(s):

Autoimmune Disease ◽

Inflammatory Responses ◽

Type I Interferon ◽

Acute Infection ◽

Murine Cytomegalovirus ◽

Type I ◽

Virus Infections ◽

Mcmv Infection ◽

Interferon Responses ◽

Deficient Mice

Siglec-H is a DAP12-associated receptor on plasmacytoid dendritic cells (pDCs) and microglia. Siglec-H inhibits TLR9-induced IFN-α production by pDCs. Previously, it was found that Siglec-H-deficient mice develop a lupus-like severe autoimmune disease after persistent murine cytomegalovirus (mCMV) infection. This was due to enhanced type I interferon responses, including IFN-α. Here we examined, whether other virus infections can also induce autoimmunity in Siglec-H-deficient mice. To this end we infected Siglec-H-deficient mice with influenza virus or with Lymphocytic Choriomeningitis virus (LCMV) clone 13. With both types of viruses we did not observe induction of autoimmune disease in Siglec-H-deficient mice. This can be explained by the fact that both types of viruses are ssRNA viruses that engage TLR7, rather than TLR9. Also, Influenza causes an acute infection that is rapidly cleared and the chronicity of LCMV clone 13 may not be sufficient and may rather suppress pDC functions. Siglec-H inhibited exclusively TLR-9 driven type I interferon responses, but did not affect type II or type III interferon production by pDCs. Siglec-H-deficient pDCs showed impaired Hck expression, which is a Src-family kinase expressed in myeloid cells, and downmodulation of the chemokine receptor CCR9, that has important functions for pDCs. Accordingly, Siglec-H-deficient pDCs showed impaired migration towards the CCR9 ligand CCL25. Furthermore, autoimmune-related genes such as Klk1 and DNase1l3 are downregulated in Siglec-H-deficient pDCs as well. From these findings we conclude that Siglec-H controls TLR-9-dependent, but not TLR-7 dependent inflammatory responses after virus infections and regulates chemokine responsiveness of pDCs.

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NAA10 p.(D10G) and NAA10 p.(L11R) Variants Hamper Formation of the NatA N-Terminal Acetyltransferase Complex

International Journal of Molecular Sciences ◽

10.3390/ijms21238973 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8973

Author(s):

Nina McTiernan ◽

Christine Darbakk ◽

Rasmus Ree ◽

Thomas Arnesen

Keyword(s):

Amino Acids ◽

Functional Analysis ◽

Catalytic Activity ◽

Complex Formation ◽

Genetic Disease ◽

De Novo ◽

Disease Mechanisms ◽

Functional Consequences

The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits—the catalytic subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles independent of NatA. Several inherited and de novo NAA10 variants have been associated with genetic disease in humans. In this study, we present a functional analysis of two de novo NAA10 variants, c.29A>G p.(D10G) and c.32T>G p.(L11R), previously identified in a male and a female, respectively. Both of these neighbouring amino acids are highly conserved in NAA10. Immunoprecipitation experiments revealed that both variants hamper complex formation with NAA15 and are thus likely to impair NatA-mediated Nt-acetylation in vivo. Despite their common impact on NatA formation, in vitro Nt-acetylation assays showed that the variants had opposing impacts on NAA10 catalytic activity. While NAA10 c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity, NAA10 c.32T>G p.(L11R) displays reduced NatA activity and normal NAA10 NAT activity. This study expands the scope of research into the functional consequences of NAA10 variants and underlines the importance of understanding the diverse cellular roles of NAA10 in disease mechanisms.

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Impact of Intermediate Hyperglycemia and Diabetes on Immune Dysfunction in Tuberculosis

Clinical Infectious Diseases ◽

10.1093/cid/ciaa751 ◽

2020 ◽

Author(s):

Clare Eckold ◽

Vinod Kumar ◽

January Weiner ◽

Bachti Alisjahbana ◽

Anca-Lelia Riza ◽

...

Keyword(s):

Gene Expression ◽

Type I Interferon ◽

Interferon Response ◽

Type I ◽

Expression Change ◽

Tb Treatment ◽

Increased Risk ◽

Patients With Diabetes ◽

Transcriptomic Signature ◽

Interferon Responses

Abstract Background People with diabetes have an increased risk of developing active tuberculosis (TB) and are more likely to have poor TB-treatment outcomes, which may impact on control of TB as the prevalence of diabetes is increasing worldwide. Blood transcriptomes are altered in patients with active TB relative to healthy individuals. The effects of diabetes and intermediate hyperglycemia (IH) on this transcriptomic signature were investigated to enhance understanding of immunological susceptibility in diabetes-TB comorbidity. Methods Whole blood samples were collected from active TB patients with diabetes (glycated hemoglobin [HbA1c] ≥6.5%) or IH (HbA1c = 5.7% to <6.5%), TB-only patients, and healthy controls in 4 countries: South Africa, Romania, Indonesia, and Peru. Differential blood gene expression was determined by RNA-seq (n = 249). Results Diabetes increased the magnitude of gene expression change in the host transcriptome in TB, notably showing an increase in genes associated with innate inflammatory and decrease in adaptive immune responses. Strikingly, patients with IH and TB exhibited blood transcriptomes much more similar to patients with diabetes-TB than to patients with only TB. Both diabetes-TB and IH-TB patients had a decreased type I interferon response relative to TB-only patients. Conclusions Comorbidity in individuals with both TB and diabetes is associated with altered transcriptomes, with an expected enhanced inflammation in the presence of both conditions, but also reduced type I interferon responses in comorbid patients, suggesting an unexpected uncoupling of the TB transcriptome phenotype. These immunological dysfunctions are also present in individuals with IH, showing that altered immunity to TB may also be present in this group. The TB disease outcomes in individuals with IH diagnosed with TB should be investigated further.

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Ligand-dependent active-site closure revealed in the crystal structure ofMycobacterium tuberculosisMenB complexed with product analogues

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714019440 ◽

2014 ◽

Vol 70 (11) ◽

pp. 2959-2969 ◽

Cited By ~ 3

Author(s):

Haigang Song ◽

Hoi Pang Sung ◽

Yuk Sing Tse ◽

Ming Jiang ◽

Zhihong Guo

Keyword(s):

Amino Acids ◽

Conformational Changes ◽

Active Site ◽

Structural Changes ◽

Catalytic Mechanism ◽

Bulk Solution ◽

Type I ◽

Important Target ◽

New Antibiotics ◽

Carboxy Terminal

1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase catalyzes an essential intramolecular Claisen condensation in menaquinone biosynthesis and is an important target for the development of new antibiotics. This enzyme inMycobacterium tuberculosisis cofactor-free and is classified as a type II DHNA-CoA synthase, differing from type I enzymes, which rely on exogenous bicarbonate for catalysis. Its crystal structures in complex with product analogues have been determined at high resolution to reveal ligand-dependent structural changes, which include the ordering of a 27-residue active-site loop (amino acids 107–133) and the reorientation of the carboxy-terminal helix (amino acids 289–301) that forms part of the active site from the opposing subunit across the trimer–trimer interface. These structural changes result in closure of the active site to the bulk solution, which is likely to take place through an induced-fit mechanism, similar to that observed for type I DHNA-CoA synthases. These findings demonstrate that the ligand-dependent conformational changes are a conserved feature of all DHNA-CoA synthases, providing new insights into the catalytic mechanism of this essential tubercular enzyme.

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Role of NLRs in the Regulation of Type I Interferon Signaling, Host Defense and Tolerance to Inflammation

International Journal of Molecular Sciences ◽

10.3390/ijms22031301 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1301

Author(s):

Ioannis Kienes ◽

Tanja Weidl ◽

Nora Mirza ◽

Mathias Chamaillard ◽

Thomas A. Kufer

Keyword(s):

Viral Infections ◽

Type I Interferon ◽

Tissue Injury ◽

Interferon Response ◽

Type I ◽

Interferon Signaling ◽

Microbial Products ◽

Interferon Responses

Type I interferon signaling contributes to the development of innate and adaptive immune responses to either viruses, fungi, or bacteria. However, amplitude and timing of the interferon response is of utmost importance for preventing an underwhelming outcome, or tissue damage. While several pathogens evolved strategies for disturbing the quality of interferon signaling, there is growing evidence that this pathway can be regulated by several members of the Nod-like receptor (NLR) family, although the precise mechanism for most of these remains elusive. NLRs consist of a family of about 20 proteins in mammals, which are capable of sensing microbial products as well as endogenous signals related to tissue injury. Here we provide an overview of our current understanding of the function of those NLRs in type I interferon responses with a focus on viral infections. We discuss how NLR-mediated type I interferon regulation can influence the development of auto-immunity and the immune response to infection.

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MDA5 is an essential vita-PAMP sensor necessary for host resistance against Aspergillus fumigatus

10.1101/2020.07.06.182154 ◽

2020 ◽

Author(s):

Xi Wang ◽

Alayna K. Caffrey-Carr ◽

Ko-wei Liu ◽

Vanessa Espinosa ◽

Walburga Croteau ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Host Resistance ◽

Type I Interferons ◽

Interferon Response ◽

Type I ◽

List Type ◽

Key Points ◽

Rna Sensors ◽

Anti Fungal ◽

Interferon Responses

ABSTRACTRIG-I like receptors (RLR) are cytosolic RNA sensors that signal through the MAVS adaptor to activate interferon responses against viruses. Whether the RLR family has broader effects on host immunity against other pathogen families remains to be fully explored. Herein we demonstrate that MDA5/MAVS signaling was essential for host resistance against pulmonary Aspergillus fumigatus challenge through the regulation of antifungal leukocyte responses in mice. Activation of MDA5/MAVS signaling was driven by dsRNA from live A. fumigatus serving as a key vitality-sensing pattern-recognition receptor. Interestingly, induction of type I interferons after A. fumigatus challenge was only partially dependent on MDA5/MAVS signaling, whereas type III interferon expression was entirely dependent on MDA5/MAVS signaling. Ultimately, type I and III interferon signaling drove the expression of CXCL10. Furthermore, the MDA5/MAVS-dependent interferon response was critical for the induction of optimal antifungal neutrophil killing of A. fumigatus spores. In conclusion, our data broaden the role of the RLR family to include a role in regulating antifungal immunity against A. fumigatus.KEY POINTSMDA5 is essential for maintaining host resistance against Aspergillus fumigatusMDA5 serves as a critical vitality sensor after fungal challengeMDA5 is essential for IFNλ expression and anti-fungal neutrophil killing

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TF protein of Sindbis virus antagonizes host type I interferon responses in a palmitoylation-dependent manner

10.1101/2019.12.13.875781 ◽

2019 ◽

Author(s):

Rogers KJ. ◽

Jones-Burrage S. ◽

Maury W. ◽

Mukhopadhyay S.

Keyword(s):

Sindbis Virus ◽

Interferon Response ◽

Type I ◽

Dependent Manner ◽

Wild Type ◽

Particle Assembly ◽

Viral Loads ◽

Interferon Responses ◽

Deficient Mice

AbstractSindbis virus (SINV) produces the small membrane protein TF from the 6K gene via a (−1) programmed ribosomal frameshifting. While several groups have shown that TF-deficient virus exhibits reduced virulence, mechanism(s) by which this occurs remain unknown. Here, we demonstrate a role for TF in antagonizing the host interferon response. Using wild-type and type 1 interferon receptor-deficient mice and primary cells derived from these animals, we show that TF controls the induction of the host interferon responses at early times during infection. Loss of TF production leads to elevated interferon and a concurrent reduction in viral loads with a loss of pathogenicity. Palmitoylation of TF has been shown to be important for particle assembly and morphology. We find that palmitoylation of TF also contributes to the ability of TF to antagonize host interferon responses as dysregulated palmitoylation of TF reduces virulence in a manner similar to loss of TF.

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TLR7-agonist and antineoplastic MEK1/2-inhibitor combination unlocks interferon responses from macrophages

10.1101/615609 ◽

2019 ◽

Author(s):

Lei Yang ◽

Jeak Ling Ding

Keyword(s):

Combination Therapy ◽

Tumor Cells ◽

Mitogen Activated Protein Kinase ◽

Interferon Response ◽

Type I ◽

Model Combination ◽

Interferon Signature ◽

Tlr7 Agonist ◽

Novel Strategy ◽

Interferon Responses

AbstractType I interferons are a family of pleiotropic cytokines that exert anti-tumor actions directly on tumor cells and indirectly on the tumor immune microenvironment (TIME). Hitherto, therapeutic strategies aiming to garner the efficacies of interferon responses are still limited. Here we show a novel strategy that elicits an interferon signature response while targeting both tumor cells using antineoplastic mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) inhibitor and the TIME using toll-like receptor 7 (TLR7)-based immune adjuvant. The combination of MEK1/2 inhibitor and TLR7 agonist unlocked an interferon signature response unexpectedly in macrophages, which was otherwise tightly constrained by TLR7 agonist alone. Deficiency of interferon regulatory factor 1 (Irf1) completely abrogated the responses and prevented the reprogramming of activated macrophages, subduing them in an immunosuppressive state. In a murine melanoma model, combination therapy with TLR7 agonist and MEK1/2 inhibitor synergistically extended survival in wild-type but notIrf1-deficient mice. Specifically, we identified interferon response genes as favorable prognosis markers for cutaneous melanoma patients. Our findings demonstrate a novel strategy for combination therapy that targets both tumor cells and the immunosuppressive TIME through additive effects of monotherapies and synergistic interferon responses.

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Localization of the nucleolar protein NO38 in amphibian oocytes.

The Journal of Cell Biology ◽

10.1083/jcb.116.1.1 ◽

1992 ◽

Vol 116 (1) ◽

pp. 1-14 ◽

Cited By ~ 58

Author(s):

B A Peculis ◽

J G Gall

Keyword(s):

Amino Acids ◽

Germinal Vesicle ◽

Nucleolar Protein ◽

Protein Accumulation ◽

Western Blots ◽

Nuclear Localization Signals ◽

Specific Accumulation ◽

Essential Domain ◽

Carboxy Terminal

To examine the role of primary amino acid sequence in the localization of proteins within the nucleus, we studied the nucleolar protein NO38 of amphibian oocytes. We synthesized NO38 transcripts in vitro, injected them into the oocyte cytoplasm, and followed the distribution of the translation products. The injected RNA contained a short sequence encoding an epitope derived from the human c-myc protein. We used an mAb against this epitope to detect translation products from injected RNAs by Western blots and by immunofluoresent staining of cytological preparations. When full-length transcripts of NO38 were injected into oocytes, the translation products accumulated efficiently in the germinal vesicle, and a major fraction was localized in the multiple nucleoli. To identify protein domains involved in this nucleolus-specific accumulation, we prepared a series of carboxy-terminal deletions of the cDNA. Oocytes injected with RNA encoding truncated forms of NO38 were examined for altered patterns of protein accumulation. We defined a domain of about 24 amino acids near the carboxy terminus that was essential for nucleolar localization of NO38. This domain is separated by more than 70 amino acids from two putative nuclear localization signals near the middle of the molecule. Hybrid constructs were made which encoded part of Escherichia coli beta-galactosidase or pyruvate kinase fused to a long segment of NO38 containing the essential domain. Injection of RNA from these constructs showed that the essential domain was not sufficient to target the hybrid proteins to the nucleolus. We suggest that nucleolar accumulation of NO38 requires more than a single linear domain.

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