Structures of Arenaviral Nucleoproteins with Triphosphate dsRNA Reveal a Unique Mechanism of Immune Suppression

A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3′-5′ exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5′ triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

Download Full-text

The 2′-O-methylation status of a single guanosine controls transfer RNA–mediated Toll-like receptor 7 activation or inhibition

Journal of Experimental Medicine ◽

10.1084/jem.20111075 ◽

2012 ◽

Vol 209 (2) ◽

pp. 235-241 ◽

Cited By ~ 61

Author(s):

Stefanie Jöckel ◽

Gernot Nees ◽

Romy Sommer ◽

Yang Zhao ◽

Dmitry Cherkasov ◽

...

Keyword(s):

Influenza A ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Thermus Thermophilus ◽

Methylation Status ◽

Transfer Rna ◽

Type I ◽

Peripheral Blood Mononuclear ◽

Molecular Pattern

Foreign RNA serves as pathogen-associated molecular pattern (PAMP) and is a potent immune stimulator for innate immune receptors. However, the role of single bacterial RNA species in immune activation has not been characterized in detail. We analyzed the immunostimulatory potential of transfer RNA (tRNA) from different bacteria. Interestingly, bacterial tRNA induced type I interferon (IFN) and inflammatory cytokines in mouse dendritic cells (DCs) and human peripheral blood mononuclear cells (PBMCs). Cytokine production was TLR7 dependent because TLR7-deficient mouse DCs did not respond and TLR7 inhibitory oligonucleotides inhibited tRNA-mediated activation. However, not all bacterial tRNA induced IFN-α because tRNA from Escherichia coli Nissle 1917 and Thermus thermophilus were non-immunostimulatory. Of note, tRNA from an E. coli knockout strain for tRNA (Gm18)-2′-O-methyltransferase (trmH) regained immunostimulatory potential. Additionally, in vitro methylation of this immunostimulatory Gm18-negative tRNA with recombinant trmH from T. thermophilus abolished its IFN-α inducing potential. More importantly, Gm18-modified tRNA acted as TLR7 antagonist and blocked IFN-α induction of influenza A virus–infected PBMCs.

Download Full-text

Differential Immune Responses to Hemorrhagic Fever-Causing Arenaviruses

Vaccines ◽

10.3390/vaccines7040138 ◽

2019 ◽

Vol 7 (4) ◽

pp. 138 ◽

Cited By ~ 4

Author(s):

Mantlo ◽

Paessler ◽

Huang

Keyword(s):

Clinical Importance ◽

Hemorrhagic Fever ◽

Lassa Fever ◽

Innate Immune ◽

Host Immune System ◽

Lassa Virus ◽

Type I ◽

Highly Pathogenic ◽

The Family ◽

Pro Inflammatory Cytokines

The family Arenaviridae contains several pathogens of major clinical importance. The Old World (OW) arenavirus Lassa virus is endemic in West Africa and is estimated to cause up to 300,000 infections each year. The New World (NW) arenaviruses Junín and Machupo periodically cause hemorrhagic fever outbreaks in South America. While these arenaviruses are highly pathogenic in humans, recent evidence indicates that pathogenic OW and NW arenaviruses interact with the host immune system differently, which may have differential impacts on viral pathogenesis. Severe Lassa fever cases are characterized by profound immunosuppression. In contrast, pathogenic NW arenavirus infections are accompanied by elevated levels of Type I interferon and pro-inflammatory cytokines. This review aims to summarize recent findings about interactions of these pathogenic arenaviruses with the innate immune machinery and the subsequent effects on adaptive immunity, which may inform the development of vaccines and therapeutics against arenavirus infections.

Download Full-text

The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism

mBio ◽

10.1128/mbio.01872-15 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 28

Author(s):

Yi Wang ◽

Li Liu

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Driving Force ◽

M Protein ◽

Signaling Cascade ◽

Type I ◽

Toll Like Receptor ◽

Beta Interferon ◽

Molecular Pattern ◽

Pathogen Associated Molecular Pattern ◽

First Time

ABSTRACT Most of the intracellular pattern recognition receptors (PRRs) reside in either the endolysosome or the cytoplasm to sense pathogen-derived RNAs, DNAs, or synthetic analogs of double-stranded RNA (dsRNA), such as poly(I:C). However, it remains elusive whether or not a pathogen-derived protein can function as a cytosolic pathogen-associated molecular pattern (PAMP). In this study, we demonstrate that delivering the membrane gene of severe acute respiratory syndrome coronavirus (SARS-CoV) into HEK293T, HEK293ET, and immobilized murine bone marrow-derived macrophage (J2-Mφ) cells significantly upregulates beta interferon (IFN-β) production. Both NF-κB and TBK1-IRF3 signaling cascades are activated by M gene products. M protein rather than M mRNA is responsible for M-mediated IFN-β induction that is preferentially associated with the activation of the Toll-like receptor (TLR) adaptor proteins MyD88, TIRAP, and TICAM2 but not the RIG-I signaling cascade. Blocking the secretion of M protein by brefeldin A (BFA) failed to reverse the M-mediated IFN-β induction. The antagonist of both TLR2 and TLR4 did not impede M-mediated IFN-β induction, indicating that the driving force for the activation of IFN-β production was generated from inside the cells. Inhibition of TRAF3 expression by specific small interfering RNA (siRNA) did not prevent M-mediated IFN-β induction. SARS-CoV pseudovirus could induce IFN-β production in an M rather than M(V68A) dependent manner, since the valine-to-alanine alteration at residue 68 in M protein markedly inhibited IFN-β production. Overall, our study indicates for the first time that a pathogen-derived protein is able to function as a cytosolic PAMP to stimulate type I interferon production by activating a noncanonical TLR signaling cascade in a TRAF3-independent manner. IMPORTANCE Viral protein can serve as a pathogen-associated molecular pattern (PAMP) that is usually recognized by certain pathogen recognition receptors (PRRs) on the cell surface, such as Toll-like receptor 2 (TLR2) and TLR4. In this study, we demonstrate that the membrane (M) protein of SARS-CoV can directly promote the activation of both beta interferon (IFN-β) and NF-κB through a TLR-related signaling pathway independent of TRAF3. The driving force for M-mediated IFN-β production is most likely generated from inside the cells. M-mediated IFN-β induction was confirmed at the viral infection level since a point mutation at the V68 residue of M markedly inhibited SARS-CoV pseudovirally induced IFN-β production. Thus, the results indicate for the first time that SARS-CoV M protein may function as a cytosolic PAMP to stimulate IFN-β production by activating a TLR-related TRAF3-independent signaling cascade.

Download Full-text

Deep assessment of human disease-associated ribosomal RNA modifications using Nanopore direct RNA sequencing

10.1101/2021.11.10.467884 ◽

2021 ◽

Author(s):

Isabel S Naarmann-de Vries ◽

Christiane Zorbas ◽

Amina Lemsara ◽

Maja Bencun ◽

Sarah Schudy ◽

...

Keyword(s):

Rna Sequencing ◽

Clinical Samples ◽

Sequencing Data ◽

Rna Modifications ◽

Flow Cells ◽

Wide Range ◽

Catalytically Active ◽

Induced Pluripotent ◽

First Time

The catalytically active component of ribosomes, rRNA, is long studied and heavily modified. However, little is known about functional and pathological consequences of changes in human rRNA modification status. Direct RNA sequencing on the Nanopore platform enables the direct assessment of rRNA modifications. We established a targeted Nanopore direct rRNA sequencing approach and applied it to CRISPR-Cas9 engineered HCT116 cells, lacking specific enzymatic activities required to establish defined rRNA base modifications. We analyzed these sequencing data along with wild type samples and in vitro transcribed reference sequences to specifically detect changes in modification status. We show for the first time that direct RNA-sequencing is feasible on smaller, i.e. Flongle, flow cells. Our targeted approach reduces RNA input requirements, making it accessible to the analysis of limited samples such as patient derived material. The analysis of rRNA modifications during cardiomyocyte differentiation of human induced pluripotent stem cells, and of heart biopsies from cardiomyopathy patients revealed altered modifications of specific sites, among them pseudouridine, 2-O-methylation of ribose and acetylation of cytidine. Targeted direct rRNA-seq analysis with JACUSA2 opens up the possibility to analyze dynamic changes in rRNA modifications in a wide range of biological and clinical samples.

Download Full-text

Strategies to Obtain Encapsulation and Controlled Release of Pentamidine in Mesoporous Silica Nanoparticles

Pharmaceutics ◽

10.3390/pharmaceutics10040195 ◽

2018 ◽

Vol 10 (4) ◽

pp. 195 ◽

Cited By ~ 8

Author(s):

Enrico Peretti ◽

Ivana Miletto ◽

Barbara Stella ◽

Flavio Rocco ◽

Gloria Berlier ◽

...

Keyword(s):

Controlled Release ◽

Mesoporous Silica ◽

Silica Nanoparticles ◽

Mesoporous Silica Nanoparticles ◽

Type I ◽

Pentamidine Isethionate ◽

Kinetic Release ◽

First Time ◽

Severe Limit

Pentamidine (PTM), an antiprotozoal agent used in clinics as pentamidine isethionate salt (PTM-S), recently showed high potential also for the treatment of cancer and myotonic dystrophy type I. However, a severe limit to the systemic administration of PTM is represented by its nephrotoxicity, leading to the need for a system able to achieve a controlled release of the drug. In this study, mesoporous silica nanoparticles (MSNs) were employed for the first time to encapsulate PTM. PTM-S was first used for loading experiments into bare (MSN-OH) and aminopropyl, cyanopropyl and carboxypropyl-functionalized MSNs (MSN-NH2, MSN-CN and MSN-COOH respectively) but it was not adequately loaded in any MSNs. The free base of PTM (PTM-B) was then obtained from PTM-S and successfully loaded into MSNs. Specifically, MSN-COOH exhibited the highest loading capacity. In vitro evaluation of PTM-B kinetic release from the different MSNs was carried out. An influence of the functional groups in slowing the release of the drug, when compared to bare MSNs was observed. Altogether, these results demonstrate that MSN-COOH could be a promising system to achieve a controlled release of PTM.

Download Full-text

Inhibition of Different Lassa Virus Strains by Alpha and Gamma Interferons and Comparison with a Less Pathogenic Arenavirus

Journal of Virology ◽

10.1128/jvi.78.6.3162-3169.2004 ◽

2004 ◽

Vol 78 (6) ◽

pp. 3162-3169 ◽

Cited By ~ 65

Author(s):

Marcel Asper ◽

Thomas Sternsdorf ◽

Meike Hass ◽

Christian Drosten ◽

Antje Rhode ◽

...

Keyword(s):

Vero Cells ◽

Lassa Fever ◽

Lassa Virus ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

High Pathogenicity ◽

Ifn Γ ◽

Virus Strains

ABSTRACT The high pathogenicity of Lassa virus is assumed to involve resistance to the effects of interferon (IFN). We have analyzed the effects of alpha IFN (IFN-α), IFN-γ, and tumor necrosis factor alpha (TNF-α) on replication of Lassa virus compared to the related, but less pathogenic, lymphocytic choriomeningitis virus (LCMV). Three low-passage Lassa virus strains (AV, NL, and CSF), isolated from humans with mild to fulminant Lassa fever, were tested. Lassa virus replication was inhibited by IFN-α and IFN-γ, but not TNF-α, in Huh7 and Vero cells. The degree of IFN sensitivity of a Lassa virus isolate did not correlate with disease severity in human patients. Furthermore, cytokine effects observed for Lassa virus and LCMV (strains CH-5692, Armstrong, and WE) were similar. To address the mechanisms involved in the IFN effect, we used cell lines in which overexpression of IFN-stimulated proteins promyelocytic leukemia protein (PML) and Sp100 could be induced. Both proteins reside in PML bodies, a cellular target of the LCMV and Lassa virus Z proteins. Overexpression of PML or Sp100 did not affect replication of either virus. This, together with the previous finding that PML knockout facilitates LCMV replication in vitro and in vivo (M. Djavani, J. Rodas, I. S. Lukashevich, D. Horejsh, P. P. Pandolfi, K. L. Borden, and M. S. Salvato, J. Virol. 75:6204-6208, 2001; W. V. Bonilla, D. D. Pinschewer, P. Klenerman, V. Rousson, M. Gaboli, P. P. Pandolfi, R. M. Zinkernagel, M. S. Salvato, and H. Hengartner, J. Virol. 76:3810-3818, 2002), describes PML as a mediator within the antiviral pathway rather than as a direct effector protein. In conclusion, the high pathogenicity of Lassa virus compared to LCMV is probably not due to increased resistance to the effects of IFN-α or IFN-γ. Both cytokines inhibit replication which is relevant for the design of antiviral strategies against Lassa fever with the aim of enhancing the IFN response.

Download Full-text

Rad23 Promotes the Targeting of Proteolytic Substrates to the Proteasome

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4902-4913.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4902-4913 ◽

Cited By ~ 214

Author(s):

Li Chen ◽

Kiran Madura

Keyword(s):

Cellular Proteins ◽

Regulatory Factor ◽

High Level Expression ◽

Ubiquitinated Proteins ◽

Efficient Delivery ◽

Catalytically Active ◽

High Level ◽

First Time

ABSTRACT Rad23 contains a ubiquitin-like domain (UbLR23) that interacts with catalytically active proteasomes and two ubiquitin (Ub)-associated (UBA) sequences that bind Ub. The UBA domains can bind Ub in vitro, although the significance of this interaction in vivo is poorly understood. Rad23 can interfere with the assembly of multi-Ub chains in vitro, and high-level expression caused stabilization of proteolytic substrates in vivo. We report here that Rad23 interacts with ubiquitinated cellular proteins through the synergistic action of its UBA domains. Rad23 plays an overlapping role with Rpn10, a proteasome-associated multi-Ub chain binding protein. Mutations in the UBA domains prevent efficient interaction with ubiquitinated proteins and result in poor suppression of the growth and proteolytic defects of a rad23Δ rpn10Δ mutant. High-level expression of Rad23 revealed, for the first time, an interaction between ubiquitinated proteins and the proteasome. This increase was not observed in rpn10Δ mutants, suggesting that Rpn10 participates in the recognition of proteolytic substrates that are delivered by Rad23. Overexpression of UbLR23 caused stabilization of a model substrate, indicating that an unregulated UbLR23-proteasome interaction can interfere with the efficient delivery of proteolytic substrates by Rad23. Because the suppression of a rad23Δ rpn10Δ mutant phenotype required both UbLR23 and UBA domains, our findings support the hypothesis that Rad23 encodes a novel regulatory factor that translocates ubiquitinated substrates to the proteasome.

Download Full-text

Immunoinformatics-Guided Designing of Peptide Vaccine against Lassa Virus with Dynamic and Immune Simulation Studies

10.20944/preprints201909.0076.v2 ◽

2019 ◽

Cited By ~ 1

Author(s):

Sifat Bin Sayed ◽

Zulkar Nain ◽

Faruq Abdullah ◽

Md. Shakil Ahmed khan ◽

Zahurul Haque ◽

...

Keyword(s):

Peptide Vaccine ◽

Lassa Fever ◽

Cell Mediated Immunity ◽

Lassa Virus ◽

Adequate Treatment ◽

Viral Haemorrhagic Fever ◽

Worldwide Population ◽

The Stability

Lassa virus (LASV) is responsible for a type of acute viral haemorrhagic fever referred to as Lassa fever. Lack of adequate treatment and preventive measures against LASV resulted in a high mortality rate in its endemic regions. In this study, a multi-epitope vaccine was designed using immunoinformatics as a prophylactic agent against the virus. Following a rigorous assessment, the vaccine was built using T-cell (NCTL=8 and NHTL=6) and B-cell (NLBL=4) epitopes from each LASV-derived protein with suitable linkers and adjuvant. The physicochemistry, immunogenic potency and safeness of the designed vaccine (~68 kDa) were assessed. In addition, chosen CTL and HTL epitopes of our vaccine showed 97.37% worldwide population coverage. Besides, disulphide engineering also improved the stability of the chimeric vaccine. Molecular docking of our vaccine protein with toll-like receptor (TLR2) showed binding efficiency followed by dynamic simulation for stable interaction. Furthermore, higher levels of cell-mediated immunity and rapid antigen clearance were suggested by immune simulation and repeated-exposure simulation, respectively. Finally, the optimized codons were used in in silico cloning to ensure higher expression within E. coli K12 bacterium. With further assessment both in vitro and in vivo, we believe that our proposed peptide-vaccine would be potential immunogen against Lassa fever.

Download Full-text

Septata intestinalis frequently isolated from stool of AIDS patients with a new cultivation method

Parasitology ◽

10.1017/s0031182000078318 ◽

1994 ◽

Vol 109 (3) ◽

pp. 281-289 ◽

Cited By ~ 41

Author(s):

T. Van Gool ◽

E. U. Canning ◽

H. Gilis ◽

M. A. Van Den Bergh Weerman ◽

J. K. M. Eeftinck Schattenkerk ◽

...

Keyword(s):

Intestinal Parasites ◽

Original Description ◽

Enterocytozoon Bieneusi ◽

Type I ◽

Aids Patients ◽

Cultivation System ◽

Antibiotic Solution ◽

Stool Samples ◽

First Time

SummaryTwo species of microsporidia, Enterocytozoon bieneusi and Septata intestinalis have been reported as intestinal parasites of AIDS patients. In attempts to establish E. bieneusi in vitro, spores were concentrated from stool samples from 4 AIDS patients with biopsy-proven E. bieneusi infections. After sterilization of the concentrate in antibiotic solution, the spores were added to monolayers of RK13 cells grown on the membranes of Transwells. Cultures were established from 7 stool samples from the 4 patients but in every case the species established was S. intestinalis not E. bieneusi. On retrospective examination of the stools, a very small number of spores of a size comparable to that of S. intestinalis was found but this species was not detected in biopsies. Typical septate vacuoles containing Type I tubules were observed in vitro but in contrast to the original description, meronts were intravacuolar and sporogony was mainly disporoblastic. The cultivation system, used for the first time for microsporidia, revealed the presence of unsuspected S. intestinalis infections and indicates that this species may be much more common than hitherto suspected. S. intestinalis has not previously been cultured.

Download Full-text