Evidence for in vitro and in vivo autocatalytic processingof the primary translation product of beet necrotic yellowvein virus RNA 1 by a papain-like proteinase

1997 ◽  
Vol 142 (5) ◽  
pp. 1051-1058 ◽  
Author(s):  
A. Hehn ◽  
C. Fritsch ◽  
K. E. Richards ◽  
H. Guilley ◽  
G. Jonard
Keyword(s):  
Translation Product ◽  
Primary Translation Product ◽  
Virus Rna

Elevated in vitro translation of a 25-kDa protein in renal cell carcinoma

1991 ◽  
Vol 69 (8) ◽  
pp. 561-565 ◽  
Author(s):  
Gilles Paradis ◽  
Josée Gaudreau ◽  
Gilles Frenette ◽  
Michel Thabet ◽  
Roland R. Tremblay ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Normal Tissue ◽  
Sodium Dodecyl ◽  
In Vitro Translation ◽  
Translation Product ◽  
Primary Translation Product ◽  
Rabbit Reticulocyte Lysate System

As a first step in understanding the changes in protein synthesis that occur in renal cell carcinoma, we have prepared poly(A)+ RNA from surgically removed tumors and from their normal tissue counterpart. These RNAs were then translated in vitro in the rabbit reticulocyte lysate system and the synthesized labeled polypeptides were separated by one- and two-dimensional gel electrophoresis. A major 25-kDa primary translation product was observed with all renal cell carcinomas. The synthesis of this protein was barely detectable with the RNA from normal tissue adjacent to the tumor. To determine if this protein could be further processed (removal of signal peptide and (or) core glycosylation), canine pancreatic microsomal membranes were added to the system. This addition resulted in the formation of a vertical row of three additional spots, with the same isoelectric point as the primary translation product and with molecular masses ranging from 27 to 31 kDa. The 31-kDa protein was retained on Concanavalin A. After digestion with endoglycosidase H, it was no longer visible on sodium dodecyl sulfate gels and a new 27-kDa band was generated suggesting that the mature protein was indeed a glycoprotein. Future experiments will be aimed at identifying this protein and examining its potential value as a marker of renal cell carcinoma.Key words: renal cancer, post-translational modifications, glycosylation, tumor markers.

scholarly journals Studies of translatable mRNA for rabbit C-reactive protein

1985 ◽  
Vol 227 (3) ◽  
pp. 759-765 ◽  
Author(s):  
D Samols ◽  
S S MacIntyre ◽  
I Kushner
Keyword(s):  
Acute Phase ◽  
Acute Phase Response ◽  
Phase Response ◽  
Translation Product ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Primary Translation Product ◽  
Free Translation ◽  
Translatable Mrna

C-reactive protein (CRP) mRNA was assayed by cell-free translation of poly(A)-containing liver RNA isolated both from rabbits stimulated to undergo the acute-phase response and from unstimulated control rabbits. No CRP-related translation products were identified until the denaturant methylmercury hydroxide (CH3HgOH) was added to the RNA before cell-free translation. In the presence of the denaturant, a 24000-Da translation product was synthesized which was immunochemically identifiable as the CRP primary translation product. It is likely that rabbit CRP mRNA can form a stable intramolecular duplex which interferes with its translatability in vitro. The 24000-Da CH3HgOH-facilitated cell-free translation product was not detected in poly(A)-containing liver RNA from unstimulated animals, indicating that the concentration of translatable CRP mRNA was dramatically induced during the acute-phase response. On the basis of absorption experiments, the 24000-Da CRP primary translation product was immunochemically more closely related to denatured CRP than to native CRP.

scholarly journals Mutations in the N-Terminal Region of Influenza Virus PB2 Protein Affect Virus RNA Replication but Not Transcription

2003 ◽  
Vol 77 (9) ◽  
pp. 5098-5108 ◽  
Author(s):  
Pablo Gastaminza ◽  
Beatriz Perales ◽  
Ana M. Falcón ◽  
Juan Ortín
Keyword(s):  
Influenza Virus ◽  
Rna Replication ◽  
Terminal Region ◽  
Viral Rna ◽  
Mrna Accumulation ◽  
Virus Rna ◽  
Transcription In Vitro ◽  
Type Mutant

ABSTRACT PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.

scholarly journals Plant Microrna Potential in Targeting COVID-19 Genome Offering Efficient Antiviral Mirna-based Therapies

2021 ◽  
Author(s):  
Behzad Hajieghrari ◽  
Sara Rahmanian-Koshkaki
Keyword(s):  
Mirna Target ◽  
Protein Translation ◽  
Plant Mirnas ◽  
Novel Therapy ◽  
Great Chance ◽  
Virus Rna ◽  
Cell Gene Expression ◽  
Target Binding

Abstract In 2019, the seventh member of the Coronaviridae named severe acute respiratory coronavirus II (or COVID-19) emerged in Wuhan, China. It could rapidly turn into a global pandemic. Therefore, searching for a natural/ novel therapy is urgently essential to prevent disease distribution. Based on the principle of cross-kingdom interaction, plants are a candidate as a potential source of exogenous miRNAs that can efficiently affect the host cell gene expression by promote target mRNA degradation or repress protein translation, and, or virus RNA translation/replication. The possibility of taking up the diet-derived plant-originated/artificially miRNA(s) in addition to their biological role in the host provides a pivotal clue for the study of synthesized/artificially expressed active therapeutic plant-originated miRNA(s) to promote antiviral activity. In this study, plant miRNAs, which can potentially interact with the COVID-19 genome within the 3’-UTR region and prompt antiviral function, were searched using bioinformatics approaches. RNAHybrid, RNA22, and STarMir miRNA/target detection tools were served for the possible plant miRNA/target recognition on the 3’-UTR flanking region of the COVID-19 genome by different algorithms. The RNAHybrid algorithm resulted in 63 plant miRNAs having hybridization energy with less or equal to -25 kcal/mol. They interact with diverse classes of miRNA/target binding patterns. However, each RNA22 and STarMir tools identified eight probable miRNA/target interaction candidates, in which pvu-miR159a.2 and sbi-miR5387b detected by both the RNA22 and STarMir tools at the same position. For us, they are suitable plant-derived miRNA candidates, which have the great chance of targeting the COVID-19 genome in the 3’-UTR region in vitro to induce the virus degradation and translational repression and for antiviral miRNA-based therapies without any side effects in vivo.

scholarly journals Déjà vu: Stimulating Open Drug Discovery for SARS-CoV-2

2020 ◽  
Author(s):  
Sean Ekins ◽  
Melina Mottin ◽  
Paulo R. P. S. Ramos ◽  
Bruna K. P. Sousa ◽  
Bruno Junior Neves ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Ebola Virus ◽  
Antiviral Drug ◽  
Open Science ◽  
Virus Rna ◽  
Deja Vu ◽  
Déjà Vu ◽  
Approved Drugs

In the past decade we have seen two major Ebola virus outbreaks in Africa, the Zika virus in Brazil and the current outbreak of coronavirus disease which has been named "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2). There is a strong sense of Déjà vu as the world is caught flat footed without effective treatments to administer to patients. Our team has been actively involved in several small molecule drug discovery efforts for the preceding virus outbreaks. In 2014 we used machine learning to identify 3 new molecules to test for the Ebola virus and these were subsequently shown to be active in vitro and in vivo. We have also been involved in open science approaches that leverage the community to help. In 2016 we launched the OpenZika project as an IBM World Community Grid Project that used distributed computing power of volunteers to dock large numbers of compounds into Zika and related flavivirus targets. This led us into several collaborations in which we validated computational predictions in vitro. With both of these initiatives there was some knowledge of the virus, many compounds had already been tested in the case of Ebola, whereas for Zika initially all we had was the virus RNA sequence. In the current SARS-CoV-2 outbreak, this was a completely new virus and the scientists in China and elsewhere have started from scratch. In the space of a few weeks since the outbreak is acknowledged to have started, there are now compounds suggested as active in vitro and molecules repurposed in clinical trials. While this has been impressive, we propose there may still be gaps in our approach to drug discovery for such outbreaks. There is an opportunity to repurpose additional approved drugs for this virus and we now suggest how these might be identified leveraging prior work on MERS-CoV, SARS-CoV and other viruses. We also describe some of the immense challenges and limitations of the open antiviral drug discovery approaches we have been involved in.

scholarly journals High SARS-CoV-2 load in the nasopharynx of patients with a mild form of COVID-19 is associated with clinical deterioration regardless of the hydroxychloroquine administration

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246396
Author(s):  
Alexey Komissarov ◽  
Ivan Molodtsov ◽  
Oxana Ivanova ◽  
Elena Maryukhnich ◽  
Svetlana Kudryavtseva ◽  
...  
Keyword(s):  
Risk Factor ◽  
Pharmacological Therapy ◽  
Control Group ◽  
Mild Form ◽  
Treatment Protocols ◽  
Mild Disease ◽  
Viral Shedding ◽  
Virus Rna

Because of the constantly growing numbers of COVID-19 infections and deaths, attempts were undertaken to find drugs with anti-SARS-CoV-2 activity among ones already approved for other pathologies. In the framework of such attempts, in a number of in vitro, as well as in vivo, models it was shown that hydroxychloroquine (HCQ) has an effect against SARS-CoV-2. While there were not enough clinical data to support the use of HCQ, several countries including Russia have included HCQ in treatment protocols for infected patients and for prophylaxis. In the current non-randomized, observational study we evaluated the SARS-CoV-2 RNA in nasopharynx swabs from infected patients 7–10 days post symptoms with clinically mild disease and compared the viral RNA load dynamics between patients receiving HCQ (200 mg twice per day according to the Ministry of Health of Russian Federation treatment instructions, n = 33) and a control group without antiviral pharmacological therapy (n = 12). We found a statistically significant relationship between maximal RNA quantity and deterioration of patients’ medical conditions, and as well we confirmed arterial hypertension to be a risk factor for people with COVID-19. However, we showed that at the dose used in the study HCQ therapy neither shortened the viral shedding period nor reduced the virus RNA load.

scholarly journals Mechanism of Action of T-705 against Influenza Virus

2005 ◽  
Vol 49 (3) ◽  
pp. 981-986 ◽  
Author(s):  
Yousuke Furuta ◽  
Kazumi Takahashi ◽  
Masako Kuno-Maekawa ◽  
Hidehiro Sangawa ◽  
Sayuri Uehara ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Mdck Cells ◽  
Specific Inhibitor ◽  
Polymerase Activity ◽  
Virus Activity ◽  
Virus Rna ◽  
Cellular Dna

ABSTRACT T-705, a substituted pyrazine compound, has been found to exhibit potent anti-influenza virus activity in vitro and in vivo. In a time-of-addition study, it was indicated that T-705 targeted an early to middle stage of the viral replication cycle but had no effect on the adsorption or release stage. The anti-influenza virus activity of T-705 was attenuated by addition of purines and purine nucleosides, including adenosine, guanosine, inosine, and hypoxanthine, whereas pyrimidines did not affect its activity. T-705-4-ribofuranosyl-5′-triphosphate (T-705RTP) and T-705-4-ribofuranosyl-5′-monophosphate (T-705RMP) were detected in MDCK cells treated with T-705. T-705RTP inhibited influenza virus RNA polymerase activity in a dose-dependent and a GTP-competitive manner. Unlike ribavirin, T-705 did not have an influence on cellular DNA or RNA synthesis. Inhibition of cellular IMP dehydrogenase by T-705RMP was about 150-fold weaker than that by ribavirin monophosphate, indicating the specificity of the anti-influenza virus activity and lower level of cytotoxicity of T-705. These results suggest that T-705RTP, which is generated in infected cells, may function as a specific inhibitor of influenza virus RNA polymerase and contributes to the selective anti-influenza virus activity of T-705.

In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas

1986 ◽  
Vol 6 (9) ◽  
pp. 827-834 ◽  
Author(s):  
Ernst Bause ◽  
Roland Günther ◽  
Jürgen Schweden ◽  
Ulrich Tillmann
Keyword(s):  
Molecular Mass ◽  
Consensus Sequence ◽  
Proteolytic Processing ◽  
Translation System ◽  
Translation Product ◽  
Glycosidase Inhibitors ◽  
Glucosidase Ii ◽  
Primary Translation Product ◽  
Dog Pancreas

When programmed with yeast prepro-α-factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp-α-Fcyt, 21 kDa) and a membrane-specific and multiply glycosylated e-factor precursor (pp-α-F3, 27.5 kDa). Glycosylation of the membrane specific pp-α-F3 species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp-α-F0), whereas the primary translation product pp-α-F cyt is not affected. Likewise, only the glycosylated pp-α-F3 structure is digested by Endo H yielding a polypeptide with a molecular mass between PP-α-F0 and pp-α-F cyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp-α-F cyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp-α-F3 polypeptide indicates that its oligosaccharide chains are processed to presumbly Man9-GlcNAc2 structures under the in vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence. In addition, several arguments suggest that both trimming enzymes are located in the lumen of the microsomal vesicles derived from endoplasmic reticulum membranes.

Expression of Alfalfa Mosaic virus RNA 4 cDNA transcripts in Vitro and in Vivo

Virology ◽  
1985 ◽  
Vol 146 (2) ◽  
pp. 177-187 ◽  
Author(s):  
L. Sue Loesch-Fries ◽  
Nancy P. Jarvis ◽  
Karen J. Krahn ◽  
Steven E. Nelson ◽  
Timothy C. Hall
Keyword(s):  
Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
Virus Rna
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