Cultivation of Cells in a Physiological Plasmax Medium Increases Mitochondrial Respiratory Capacity and Reduces Replication Levels of RNA Viruses

Changes in metabolic pathways are often associated with the development of various pathologies including cancer, inflammatory diseases, obesity and metabolic syndrome. Identification of the particular metabolic events that are dysregulated may yield strategies for pharmacologic intervention. However, such studies are hampered by the use of classic cell media that do not reflect the metabolite composition that exists in blood plasma and which cause non-physiological adaptations in cultured cells. In recent years two groups presented media that aim to reflect the composition of human plasma, namely human plasma-like medium (HPLM) and Plasmax. Here we describe that, in four different mammalian cell lines, Plasmax enhances mitochondrial respiration. This is associated with the formation of vast mitochondrial networks and enhanced production of reactive oxygen species (ROS). Interestingly, cells cultivated in Plasmax displayed significantly less lysosomes than when any standard media were used. Finally, cells cultivated in Plasmax support replication of various RNA viruses, such as hepatitis C virus (HCV) influenza A virus (IAV), severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and several others, albeit at lower levels and with delayed kinetics. In conclusion, studies of metabolism in the context of viral infections, especially those concerning mitochondria, lysosomes, or redox systems, should be performed in Plasmax medium.

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SP-A and SP-D: Dual Functioning Immune Molecules With Antiviral and Immunomodulatory Properties

Frontiers in Immunology ◽

10.3389/fimmu.2020.622598 ◽

2021 ◽

Vol 11 ◽

Author(s):

Alastair Watson ◽

Jens Madsen ◽

Howard William Clark

Keyword(s):

Viral Infection ◽

Influenza A ◽

Viral Infections ◽

Inflammatory Responses ◽

Therapeutic Potential ◽

Inflammatory Diseases ◽

Chronic Obstructive ◽

Cell Functions ◽

Increased Risk ◽

Syncytial Virus

Surfactant proteins A (SP-A) and D (SP-D) are soluble innate immune molecules which maintain lung homeostasis through their dual roles as anti-infectious and immunomodulatory agents. SP-A and SP-D bind numerous viruses including influenza A virus, respiratory syncytial virus (RSV) and human immunodeficiency virus (HIV), enhancing their clearance from mucosal points of entry and modulating the inflammatory response. They also have diverse roles in mediating innate and adaptive cell functions and in clearing apoptotic cells, allergens and other noxious particles. Here, we review how the properties of these first line defense molecules modulate inflammatory responses, as well as host-mediated immunopathology in response to viral infections. Since SP-A and SP-D are known to offer protection from viral and other infections, if their levels are decreased in some disease states as they are in severe asthma and chronic obstructive pulmonary disease (COPD), this may confer an increased risk of viral infection and exacerbations of disease. Recombinant molecules of SP-A and SP-D could be useful in both blocking respiratory viral infection while also modulating the immune system to prevent excessive inflammatory responses seen in, for example, RSV or coronavirus disease 2019 (COVID-19). Recombinant SP-A and SP-D could have therapeutic potential in neutralizing both current and future strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus as well as modulating the inflammation-mediated pathology associated with COVID-19. A recombinant fragment of human (rfh)SP-D has recently been shown to neutralize SARS-CoV-2. Further work investigating the potential therapeutic role of SP-A and SP-D in COVID-19 and other infectious and inflammatory diseases is indicated.

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Upstream translation initiation expands the coding capacity of segmented negative-strand RNA viruses

10.1101/795815 ◽

2019 ◽

Author(s):

Elizabeth Sloan ◽

Marta Alenquer ◽

Liliane Chung ◽

Sara Clohisey ◽

Adam M. Dinan ◽

...

Keyword(s):

Translation Initiation ◽

Influenza A ◽

Viral Infections ◽

Rna Viruses ◽

Influenza Viruses ◽

Open Reading Frames ◽

Viral Gene ◽

Influenza A Viruses ◽

Negative Strand ◽

Coding Potential

AbstractSegmented negative-strand RNA viruses (sNSVs) include the influenza viruses, the bunyaviruses, and other major pathogens of humans, other animals and plants. The genomes of these viruses are extremely short. In response to this severe genetic constraint, sNSVs use a variety of strategies to maximise their coding potential. Because the eukaryotic hosts parasitized by sNSVs can regulate gene expression through low levels of translation initiation upstream of their canonical open reading frames (ORFs), we asked whether sNSVs could use upstream translation initiation to expand their own genetic repertoires. Consistent with this hypothesis, we showed that influenza A viruses (IAVs) and bunyaviruses were capable of upstream translation initiation. Using a combination of reporter assays and viral infections, we found that upstream translation in IAVs can initiate in two unusual ways: through non-AUG initiation in virally encoded ‘untranslated’ regions, and through the appropriation of an AUG-containing leader sequence from host mRNAs through viral cap-snatching, a process we termed ‘start-snatching.’ Finally, while upstream translation of cellular genes is mainly regulatory, for sNSVs it also has the potential to create novel viral gene products. If in frame with a viral ORF, this creates N-extensions of canonical viral proteins. If not, it allows the expression of cryptic overlapping ORFs, which we found were highly conserved in IAV and widely distributed in peribunyaviruses. Thus, by exploiting their host’s capacity for upstream translation initiation, sNSVs can expand still further the coding potential of their extremely compact RNA genomes.

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Zinc finger antiviral protein (ZAP) activity in mammalian and avian hosts in CpG and UpA-mediated restriction of RNA viruses and investigation of ZAP-mediated shaping of host transcriptome compositions

10.1101/2021.11.04.467232 ◽

2021 ◽

Author(s):

Valerie Odon ◽

Steven fiddaman ◽

Adrian Smith ◽

Peter Simmonds

Keyword(s):

Cell Lines ◽

Zinc Finger ◽

Influenza A ◽

Rna Viruses ◽

Rna Virus ◽

Virus Genome ◽

Antiviral Protein ◽

Cpg Dinucleotides ◽

Antiviral State ◽

Mammalian Cell Lines

The ability of zinc finger antiviral protein (ZAP) to recognise and respond to RNA virus sequences with elevated frequencies of CpG dinucleotides has been proposed as a functional part of the vertebrate innate immune antiviral response. It has been further proposed that ZAP activity shapes compositions of cytoplasmic mRNA sequences to avoid self-recognition, particularly mRNAs for interferons (IFNs) and IFN-stimulated genes highly expressed when ZAP is upregulated during the antiviral state. We investigated the ZAP functional activity in different species of mammals and birds, and potential downstream effects of differences in CpG and UpA dinucleotide representations in host transcriptomes and in RNA viruses that infect them. Cell lines from different bird orders showed variability in restriction of influenza A virus and echovirus 7 replicons with elevated CpG frequencies and none restricted UpA-high mutants, in marked contrast to mammalian cell lines. Given this variability, we compared CpG and UpA representation in coding regions of ISGs and IFNs with the total cellular transcriptome to determine whether differences in ZAP activity shaped dinucleotide compositions of highly expressed genes during the antiviral state. While type 1 IFN genes typically showed often profound suppression of CpG and UpA frequencies, there was no over-suppression of CpGs or UpAs in ISGs in any species, irrespective of underlying ZAP activity. Similarly, mammalian and avian RNA virus genome sequences were compositionally equivalent as were IAV serotypes recovered from ducks, chickens and humans. Overall, we found no evidence for host variability in ZAP function impacting compositions of antiviral genes.

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TRIM25 inhibits Influenza A infection by destabilising its mRNA and is redundant for the RIG-I pathway.

10.1101/2021.09.13.460052 ◽

2021 ◽

Author(s):

Nila Roy Choudhury ◽

Gregory Heikel ◽

Ivan Trus ◽

Rute Maria Dos Santos Pinto ◽

Maryia Trubitsyna ◽

...

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Ubiquitin Ligase ◽

Influenza A ◽

Rna Binding ◽

Cultured Cells ◽

Rna Viruses ◽

E3 Ubiquitin Ligase ◽

Innate Immune

The E3 ubiquitin ligase TRIM25 is a key factor in the innate immune response to RNA viruses. TRIM25 has been shown to play a role in the retinoic-acid-inducible gene-1 (RIG-I) pathway, which triggers expression of type 1 interferons upon viral infection. We and others have recently shown that TRIM25 is an RNA-binding protein, however not much is known about the RNA-binding roles of TRIM25 in the innate immune response to RNA viruses. Here, we demonstrate that influenza A virus (IAV A/PR/8/34_NS1(R38K41A)) infection is inhibited by TRIM25. Surprisingly, host RNA-binding deficient mutant TRIM25ΔRBD and TRIM25ΔRING, which lack E3 ubiquitin ligase activity rescued IAV inhibition in TRIM25 knock-out cells. Furthermore, we show that in human cultured cells activation of the RIG-I/interferon type 1 pathway mediated by an IAV-derived 5′-triphosphate RNA does not require TRIM25 activity. Additionally, knocking out TRIM25 does not affect the activity of the IAV polymerase. We present new evidence that TRIM25 restricts IAV by directly binding to and destabilising its mRNAs. Finally, we show that direct tethering of TRIM25 to RNA is sufficient to downregulate the targeted RNA. In summary, our results uncover a novel mechanism that TRIM25 uses to inhibit IAV infection and regulate RNA metabolism.

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The Role of microRNAs in the Viral Infections

Current Pharmaceutical Design ◽

10.2174/1381612825666190110161034 ◽

2019 ◽

Vol 24 (39) ◽

pp. 4659-4667 ◽

Cited By ~ 4

Author(s):

Mona Fani ◽

Milad Zandi ◽

Majid Rezayi ◽

Nastaran Khodadad ◽

Hadis Langari ◽

...

Keyword(s):

Viral Infections ◽

Rna Viruses ◽

Regulation Of Gene Expression ◽

Molecular Structures ◽

Main Function ◽

Cellular Functions ◽

Viral Genes ◽

Non Coding Rnas ◽

Post Transcriptional Regulation

MicroRNAs (miRNAs) are non-coding RNAs with 19 to 24 nucleotides which are evolutionally conserved. MicroRNAs play a regulatory role in many cellular functions such as immune mechanisms, apoptosis, and tumorigenesis. The main function of miRNAs is the post-transcriptional regulation of gene expression via mRNA degradation or inhibition of translation. In fact, many of them act as an oncogene or tumor suppressor. These molecular structures participate in many physiological and pathological processes of the cell. The virus can also produce them for developing its pathogenic processes. It was initially thought that viruses without nuclear replication cycle such as Poxviridae and RNA viruses can not code miRNA, but recently, it has been proven that RNA viruses can also produce miRNA. The aim of this articles is to describe viral miRNAs biogenesis and their effects on cellular and viral genes.

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Antimalarial drugs—are they beneficial in rheumatic and viral diseases?—considerations in COVID-19 pandemic

Clinical Rheumatology ◽

10.1007/s10067-021-05805-5 ◽

2021 ◽

Author(s):

Bogna Grygiel-Górniak

Keyword(s):

Connective Tissue ◽

Viral Infections ◽

Current Knowledge ◽

Inflammatory Diseases ◽

Antiviral Drugs ◽

In Vivo Studies ◽

Viral Diseases ◽

Cardiac Complications

AbstractThe majority of the medical fraternity is continuously involved in finding new therapeutic schemes, including antimalarial medications (AMDs), which can be useful in combating the 2019-nCoV: coronavirus disease (COVID-19). For many decades, AMDs have been widely used in the treatment of malaria and various other anti-inflammatory diseases, particularly to treat autoimmune disorders of the connective tissue. The review comprises in vitro and in vivo studies, original studies, clinical trials, and consensus reports for the analysis, which were available in medical databases (e.g., PubMed). This manuscript summarizes the current knowledge about chloroquine (CQ)/hydroxychloroquine (HCQ) and shows the difference between their use, activity, recommendation, doses, and adverse effects on two groups of patients: those with rheumatic and viral diseases (including COVID-19). In the case of connective tissue disorders, AMDs are prescribed for a prolonged duration in small doses, and their effect is observed after few weeks, whereas in the case of viral infections, they are prescribed in larger doses for a short duration to achieve a quick saturation effect. In rheumatic diseases, AMDs are well tolerated, and their side effects are rare. However, in some viral diseases, the effect of AMDs is questionable or not so noticeable as suggested during the initial prognosis. They are mainly used as an additive therapy to antiviral drugs, but recent studies have shown that AMDs can diminish the efficacy of some antiviral drugs and may cause respiratory, kidney, liver, and cardiac complications.

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Disease severity-specific neutrophil signatures in blood transcriptomes stratify COVID-19 patients

Genome Medicine ◽

10.1186/s13073-020-00823-5 ◽

2021 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Anna C. Aschenbrenner ◽

◽

Maria Mouktaroudi ◽

Benjamin Krämer ◽

Marie Oestreich ◽

...

Keyword(s):

Immune System ◽

Disease Severity ◽

Whole Blood ◽

Viral Infections ◽

Inflammatory Diseases ◽

Target Prediction ◽

Data Driven ◽

Host Responses ◽

Drug Candidates ◽

Drug Target Prediction

Abstract Background The SARS-CoV-2 pandemic is currently leading to increasing numbers of COVID-19 patients all over the world. Clinical presentations range from asymptomatic, mild respiratory tract infection, to severe cases with acute respiratory distress syndrome, respiratory failure, and death. Reports on a dysregulated immune system in the severe cases call for a better characterization and understanding of the changes in the immune system. Methods In order to dissect COVID-19-driven immune host responses, we performed RNA-seq of whole blood cell transcriptomes and granulocyte preparations from mild and severe COVID-19 patients and analyzed the data using a combination of conventional and data-driven co-expression analysis. Additionally, publicly available data was used to show the distinction from COVID-19 to other diseases. Reverse drug target prediction was used to identify known or novel drug candidates based on finding from data-driven findings. Results Here, we profiled whole blood transcriptomes of 39 COVID-19 patients and 10 control donors enabling a data-driven stratification based on molecular phenotype. Neutrophil activation-associated signatures were prominently enriched in severe patient groups, which was corroborated in whole blood transcriptomes from an independent second cohort of 30 as well as in granulocyte samples from a third cohort of 16 COVID-19 patients (44 samples). Comparison of COVID-19 blood transcriptomes with those of a collection of over 3100 samples derived from 12 different viral infections, inflammatory diseases, and independent control samples revealed highly specific transcriptome signatures for COVID-19. Further, stratified transcriptomes predicted patient subgroup-specific drug candidates targeting the dysregulated systemic immune response of the host. Conclusions Our study provides novel insights in the distinct molecular subgroups or phenotypes that are not simply explained by clinical parameters. We show that whole blood transcriptomes are extremely informative for COVID-19 since they capture granulocytes which are major drivers of disease severity.

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Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

Scientific Reports ◽

10.1038/s41598-021-84551-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alessandro T. Caputo ◽

Oliver M. Eder ◽

Hana Bereznakova ◽

Heleen Pothuis ◽

Albert Ardevol ◽

...

Keyword(s):

Cell Lines ◽

Mammalian Cell ◽

Recombinant Proteins ◽

Cultured Cells ◽

Hek293 Cells ◽

Reaction Products ◽

Enzyme Form ◽

X Ray Crystallography ◽

Mammalian Cell Lines ◽

Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

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Inflammatory and Obstructive Disorders of Salivary Glands

Journal of Dental Research ◽

10.1177/00220345870660s112 ◽

1987 ◽

Vol 66 (1_suppl) ◽

pp. 675-679 ◽

Cited By ~ 6

Author(s):

A. Blitzer

Keyword(s):

Salivary Glands ◽

Fine Needle Aspiration ◽

Chronic Conditions ◽

Viral Infections ◽

Inflammatory Diseases ◽

Needle Aspiration ◽

Ct Scans ◽

Fine Needle ◽

Inflammatory Conditions ◽

Chronic Sialadenitis

Obstructive and inflammatory diseases of the salivary glands can have a congenital, traumatic, metabolic, or infectious-inflammatory cause. The acute inflammatory conditions include bacterial and viral infections, and the chronic conditions include sialoliths, strictures, chronic sialadenitis, sialectasis, and lymphoepithelial disease. The neoplastic diseases can cause obstruction and/or infection and often make the diagnosis elusive. In addition to a working knowledge of possible etiology, one needs experience with clinical examination, salivary analysis, sialography, CT scans, MRI, and fine-needle aspiration and cytology in order successfully to evaluate and manage patients with these conditions.

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