scholarly journals Proteolytic processing of potyviral proteins and polyprotein processing intermediates in insect and plant cells

2002 ◽  
Vol 83 (5) ◽  
pp. 1211-1221 ◽  
Author(s):  
Andres Merits ◽  
Minna-Liisa Rajamäki ◽  
Päivi Lindholm ◽  
Pia Runeberg-Roos ◽  
Tuija Kekarainen ◽  
...  
Keyword(s):  
Virus Replication ◽  
Time Course ◽  
Infected Plant ◽  
Plant Cells ◽  
Proteolytic Processing ◽  
High Rate ◽  
Detectable Effect ◽  
Virus Infectivity ◽  
Polyprotein Processing ◽  
Baculovirus System

Processing of the polyprotein encoded by Potato virus A (PVA; genus Potyvirus) was studied using expression of the complete PVA polyprotein or its mutants from recombinant baculoviruses in insect cells. The time-course of polyprotein processing by the main viral proteinase (NIaPro) was examined with the pulse–chase method. The sites at the P3/6K1, CI-6K2 and VPg/NIaPro junctions were processed slowly, in contrast to other proteolytic cleavage sites which were processed at a high rate. The CI-6K2 polyprotein was observed in the baculovirus system and in infected plant cells. In both cell types the majority of CI-6K2 was found in the membrane fraction, in contrast to fully processed CI. Deletion of the genomic region encoding the 6K1 protein prevented proper proteolytic separation of P3 from CI, but did not affect processing of VPg, NIaPro, NIb or CP from the polyprotein. The 6K2-encoding sequence could be removed without any detectable effect on polyprotein processing. However, deletion of either the 6K1 or 6K2 protein-encoding regions rendered PVA non-infectious. Mutations at the 6K2/VPg cleavage site reduced virus infectivity in plants, but had a less pronounced, albeit detectable, effect on proteolytic processing in the baculovirus system. The results of this study indicate that NIaPro catalyses proteolytic cleavages preferentially in cis, and that the 6K1/CI and NIb/CP sites can also be processed in trans. Both 6K peptides are indispensable for virus replication, and proteolytic separation of the 6K2 protein from the adjacent proteins by NIaPro is important for the rate of virus replication and movement.

scholarly journals Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Critical for Ebola Virus Replication in Nonhuman Primates

2007 ◽  
Vol 81 (6) ◽  
pp. 2995-2998 ◽  
Author(s):  
Gabriele Neumann ◽  
Thomas W. Geisbert ◽  
Hideki Ebihara ◽  
Joan B. Geisbert ◽  
Kathleen M. Daddario-DiCaprio ◽  
...  
Keyword(s):  
Virus Replication ◽  
Nonhuman Primates ◽  
Ebola Virus ◽  
Proteolytic Processing ◽  
Surface Glycoprotein ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Recognition Sequence ◽  
Furin Cleavage ◽  
Virus Glycoprotein

ABSTRACT Enveloped viruses often require cleavage of a surface glycoprotein by a cellular endoprotease such as furin for infectivity and virulence. Previously, we showed that Ebola virus glycoprotein does not require the furin cleavage motif for virus replication in cell culture. Here, we show that there are no appreciable differences in disease progression, hematology, serum biochemistry, virus titers, or lethality in nonhuman primates infected with an Ebola virus lacking the furin recognition sequence compared to those infected with wild-type virus. We conclude that glycoprotein cleavage by subtilisin-like endoproteases is not critical for Ebola virus infectivity and virulence in nonhuman primates.

Quantitative aspects of RNA synthesis and polyadenylation in 1-cell and 2-cell mouse embryos

Development ◽  
1983 ◽  
Vol 74 (1) ◽  
pp. 169-182
Author(s):  
Kerry B. Clegg ◽  
Lajos Pikó
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Average Length ◽  
State Level ◽  
Mouse Embryos ◽  
High Rate ◽  
Rna Sequences ◽  
Cell Stage ◽  
New Synthesis ◽  
Cell Embryo

Mouse embryos at the late 1-cell and late 2-cell stages were labelled with [3H]adenosine for periods of up to 320 min during which the specific activity of the ATP pool was constant. The time course of the molar accumulation of adenosine was calculated for tRNA, high-molecular-weight poly(A)− RNA and poly(A) tails versus internal regions of poly(A)+ RNA. Most of the adenosine incorporation into tRNA is due to turnover of the 3′-terminal AMP but some new synthesis of tRNA also appears to take place in both 1-cell and 2-cell embryos at a rate of about 0·2 pg/embryo/h. In the poly(A)- RNA fraction, an unstable component which is assumed to be heterogeneous nuclear RNA is synthesized at a high rate and accumulates at a steady-state level of about 1·5 pg/embryo in the 1-cell embryo and about 3·0 pg/embryo in the 2-cell embryo. Both 1-cell and 2-cell embryos synthesize relatively stable heterogeneous poly(A)− RNA, assumed to be mRNA, at a rate of about 0·3 pg/embryo/h; 2-cell embryos also synthesize mature ribosomal RNA at a rate of about 0·4 pg/embryo·h. Internally labelled poly(A)+ RNA is synthesized at a low rate in the 1-cell embryo, about 0·045 pg/embryo/h, but the rate increases to about 0·2 pg/embryo/h by the 2-cell stage. A striking feature of the 1-cell embryo is the high rate of synthesis of poly(A) tails, about 2·5 × 106 tails/embryo/h of an average length of (A)43, due almost entirely to cytoplasmic polyadenylation. This and other evidence suggests a turnover of the poly(A)+ RNA population in 1-cell embryos as a result of polyadenylation of new RNA sequences and degradation of some of the pre-existing poly(A)+ RNA. In the 2-cell embryo, the rate of synthesis of poly(A) tails (average length (A)93) is estimated at about 0·8 × 106tails/embryo/h and a significant fraction of poly(A) synthesis appears to be nuclear.

scholarly journals Expression and Processing of Proteins Encoded by the Saccharomyces Retrotransposon Ty5

2001 ◽  
Vol 75 (4) ◽  
pp. 1790-1797 ◽  
Author(s):  
Phillip A. Irwin ◽  
Daniel F. Voytas
Keyword(s):  
Reverse Transcriptase ◽  
Time Course ◽  
Immunoblot Analysis ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Reading Frame ◽  
C Terminus ◽  
Virus Like Particle ◽  
Ionic Detergent ◽  
Time Course Experiment

ABSTRACT Retroelements (retrotransposons and retroviruses) have two genes in common: gag, which specifies structural proteins that form a virus or virus-like particle, andpol, which specifies catalytic proteins required for replication. For many retroelements, gag andpol are present on separate reading frames. Their expression is highly regulated, and the ratio of Gag to Pol is critical for retroelement replication. The Saccharomycesretrotransposon Ty5 contains a single open reading frame, and we characterized Gag and Pol expression by generating transpositionally active Ty5 elements with epitope tags at the N terminus or C terminus or within the integrase coding region. Immunoblot analysis identified two Gag species (Gag-p27 and Gag-p37), reverse transcriptase (Pol-p59), and integrase (Pol-p80), all of which are largely insoluble in the absence of urea or ionic detergent. These proteins result from proteolytic processing of a polyprotein, because elements with mutations in the presumed active site of Ty5 protease express a single tagged protein (Gag-Pol-p182). Protease mutants are also transpositionally inactive. In a time course experiment, we monitored protein expression, proteolytic processing, and transposition of a Ty5 element with identical epitope tags at its N and C termini. Both transposition and the abundance of Gag-p27 increased over time. In contrast, the levels of Gag-p37 and reverse transcriptase peaked after ∼14 h of induction and then gradually decreased. This may be due to differences in stability of Gag-p27 relative to Gag-p37 and reverse transcriptase. The ratio of Ty5 Gag to Pol averaged 5:1 throughout the time course experiment, suggesting that differential protein stability regulates the amounts of these proteins.

scholarly journals Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus Replication

2018 ◽  
Vol 92 (8) ◽  
Author(s):  
Jonas Kjær ◽  
Graham J. Belsham
Keyword(s):  
Amino Acid ◽  
Disease Virus ◽  
Virus Replication ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Amino Acid Substitutions ◽  
2A Peptide ◽  
Polyprotein Processing ◽  
Mouth Disease Virus ◽  
Foot And Mouth

ABSTRACTFoot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational “cleavage” of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed “ribosome skipping” or “StopGo.” Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E14, S15, and N16within the 2A sequences of infectious FMDVs despite their reported “cleavage” efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P17, G18, or P19, which displayed little or no “cleavage” activityin vitro, were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E14, S15, N16, and P19resulted in partial “cleavage” of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational “cleavage.” Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient “cleavage” at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV.IMPORTANCEFoot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational “cleavage” of the FMDV polyprotein precursor at the 2A/2B junction, termed StopGo, is mediated by the short 2A peptide through a nonproteolytic mechanism which leads to release of the nascent protein and continued translation of the downstream sequence. Improved understanding of this process will not only give a better insight into how this peptide influences the FMDV replication cycle but may also assist the application of this sequence in biotechnology for the production of multiple proteins from a single mRNA. Our data show that single amino acid substitutions in the 2A peptide can have a major influence on viral protein synthesis, virus viability, and polyprotein processing. They also indicate that efficient “cleavage” at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity is not essential for the viability of FMDV.

Chios mastic gum inhibits influenza A virus replication and viral pathogenicity

2021 ◽  
Author(s):  
Dong-In Kim ◽  
Yong-Bin Cho ◽  
Younghyun Lim ◽  
So-Hee Hong ◽  
Bumsuk Hahm ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Bacterial Infections ◽  
Early Stage ◽  
Antiviral Drug ◽  
Virus Infectivity ◽  
Viral Attachment ◽  
Cellular Processes ◽  
Chios Mastic Gum

Chios mastic gum (CMG), a resin of the mastic tree (Pistacia lentiscus var. chia), has been used to treat multiple disorders caused by gastrointestinal malfunctions and bacterial infections for more than 2500 years. However, little is known about CMG’s antiviral activity. CMG is known to influence multiple cellular processes such as cell proliferation, differentiation and apoptosis. As virus replication is largely dependent on the host cellular metabolism, it is conceivable that CMG regulates virus infectivity. Therefore, in this study, we evaluated CMG’s potential as an antiviral drug to treat influenza A virus (IAV) infection. CMG treatment dramatically reduced the cytopathogenic effect and production of RNAs, proteins and infectious particles of IAV. Interestingly, CMG interfered with the early stage of the virus life cycle after viral attachment. Importantly, the administration of CMG greatly ameliorated morbidity and mortality in IAV-infected mice. The results suggest that CMG displays a potent anti-IAV activity by blocking the early stage of viral replication. Thus, mastic gum could be exploited as a novel therapeutic agent against IAV infection.

scholarly journals Blood flow to bone marrow during development of anemia or polycythemia in the rat

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 594-601 ◽  
Author(s):  
PO Iversen ◽  
G Nicolaysen ◽  
HB Benestad
Keyword(s):  
Bone Marrow ◽  
Blood Flow ◽  
Hemolytic Anemia ◽  
Time Course ◽  
Relative Increase ◽  
Detectable Effect ◽  
A Value ◽  
Microsphere Method ◽  
Radioactive Microsphere Method ◽  
Increased Blood Flow

Abstract We applied the radioactive microsphere method to follow the magnitude and time course (0 to 96 hours) of blood flow changes during development and recovery from anemia in awake rats. Blood flow was also monitored during a 96-hour period after polycythemia was induced (erythropoietin administered subcutaneously [SC]). The possible influence of innervation was also examined. After a blood loss of approximately 50% (hypovolemia), blood flow to the femoral marrow tripled within 12 hours and remained elevated for the entire 96-hour period. The relative increase in blood flow to the femoral bone was even greater. Similar findings were obtained in rats with phenylhydrazine (PHZ) hemolytic anemia (normovolemia). Denervation had no detectable effect on the increased blood flow to either marrow or bone. The augmented blood flow during hemolytic anemia was accompanied by a doubling of the oxygen consumption rate by the marrow, while the glucose uptake was not detectably altered. Erythropoietin supplements (3 x 1,000 IU/kg, SC, 6-hour intervals) increased blood flow to the marrow by approximately 25% after 48 hours, and at 72 hours the blood flow had reached a value twice that obtained under control conditions. These results indicate that blood flow to bone marrow is highly variable and hormonally and/or locally regulated. This may have practical consequences for marrow transplantation technology and for administration of drug therapy to patients with insufficient bone marrow hematopoiesis.

Parameters for growth measurement in suspension cultures of plant cells

1974 ◽  
Vol 52 (4) ◽  
pp. 903-912 ◽  
Author(s):  
Dyson Rose ◽  
S. M. Martin
Keyword(s):  
Stationary Phases ◽  
Plant Cells ◽  
Growth Period ◽  
Dry Weight ◽  
High Rate ◽  
Growth Phases ◽  
Batch Cultures ◽  
Growth Measurement ◽  
Large Increments ◽  
Uptake And Metabolism

Well-adapted cells, which had been initiated from root tissue of Ipomoea and of Daucus carota, were grown in 7.5-liter stirred-jar fermenters, and both the cells and media were analyzed for major components at intervals during the growth period. Controlled variables included the size of inoculum, the amount of sucrose, and the source and amount of nitrogen in the media.The data obtained indicate that there are two distinct growth phases in the development of batch cultures of these cell lines. The first, which we term "cytoplasmic growth phase," begins immediately upon addition of inoculum to fresh medium and is characterized by a high rate of nitrogen uptake and metabolism relative to the increase in cell dry weight. The second, or "maturation phase," is characterized by large increments in dry weight and total cell carbohydrate relative to the increments in cell nitrogen. It is suggested that the classical lag, log, and stationary phases of bacterial growth could apply only to the early hours of cytoplasmic growth, if indeed they are relevant at all.

scholarly journals Geminivirus Replication Protein Impairs SUMO Conjugation of Proliferating Cellular Nuclear Antigen at Two Acceptor Sites

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Manuel Arroyo-Mateos ◽  
Blanca Sabarit ◽  
Francesca Maio ◽  
Miguel A. Sánchez-Durán ◽  
Tabata Rosas-Díaz ◽  
...  
Keyword(s):  
Dna Repair ◽  
Posttranslational Modifications ◽  
Infected Plant ◽  
Plant Cells ◽  
Nuclear Antigen ◽  
Replication Machinery ◽  
In Planta ◽  
Content Type ◽  
Multifunctional Protein ◽  
Cellular Nuclear Antigen

ABSTRACTGeminiviruses are DNA viruses that replicate in nuclei of infected plant cells using the plant DNA replication machinery, including PCNA (proliferating cellular nuclear antigen), a cofactor that orchestrates genome duplication and maintenance by recruiting crucial players to replication forks. These viruses encode a multifunctional protein, Rep, which is essential for viral replication, induces the accumulation of the host replication machinery, and interacts with several host proteins, including PCNA and the SUMO E2 conjugation enzyme (SCE1). Posttranslational modification of PCNA by ubiquitin or SUMO plays an essential role in the switching of PCNA between interacting partners during DNA metabolism processes (e.g., replication, recombination, and repair, etc.). In yeast, PCNA sumoylation has been associated with DNA repair involving homologous recombination (HR). Previously, we reported that ectopic Rep expression results in very specific changes in the sumoylation pattern of plant cells. In this work, we show, using a reconstituted sumoylation system inEscherichia coli, that tomato PCNA is sumoylated at two residues, K254 and K164, and that coexpression of the geminivirus protein Rep suppresses sumoylation at these lysines. Finally, we confirm that PCNA is sumoylatedin plantaand that Rep also interferes with PCNA sumoylation in plant cells.IMPORTANCESUMO adducts have a key role in regulating the activity of animal and yeast PCNA on DNA repair and replication. Our work demonstrates for the first time that sumoylation of plant PCNA occurs in plant cells and that a plant virus interferes with this modification. This work marks the importance of sumoylation in allowing viral infection and replication in plants. Moreover, it constitutes a prime example of how viral proteins interfere with posttranslational modifications of selected host factors to create a proper environment for infection.

scholarly journals Identification of a Protein from Rust Fungi Transferred from Haustoria into Infected Plant Cells

2005 ◽  
Vol 18 (11) ◽  
pp. 1130-1139 ◽  
Author(s):  
Eric Kemen ◽  
Ariane C. Kemen ◽  
Maryam Rafiqi ◽  
Uta Hempel ◽  
Kurt Mendgen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Polyclonal Antibodies ◽  
Rust Fungus ◽  
Infected Plant ◽  
Plant Cells ◽  
Host Cells ◽  
Nuclear Localization Signals ◽  
Uromyces Fabae ◽  
Host Cytoplasm ◽  
Functional Efficiency

The formation of haustoria is one of the hallmarks of the interaction of obligate biotrophic fungi with their host plants. In addition to their role in nutrient uptake, it is hypothesized that haustoria are actively involved in establishing and maintaining the biotrophic relationship. We have identified a 24.3-kDa protein that exhibited a very unusual allocation. Rust transferred protein 1 from Uromyces fabae (Uf-RTP1p) was not only detected in the host parasite interface, the extrahaustorial matrix, but also inside infected plant cells by immunofluorescence and electron microscopy. Uf-RTP1p does not exhibit any similarity to sequences currently listed in the public databases. However, we identified a homolog of Uf-RTP1p in the related rust fungus Uromyces striatus (Us-RTP1p). The localization of Uf-RTP1p and Us-RTP1p inside infected plant cells was confirmed, using four independently raised polyclonal antibodies. Depending on the developmental stage of haustoria, Uf-RTP1p was found in increasing amounts in host cells, including the host nucleus. Putative nuclear localization signals (NLS) were found in the predicted RTP1p sequences. However, functional efficiency could only be verified for the Uf-RTP1p NLS by means of green fluorescent protein fusions in transformed tobacco protoplasts. Western blot analysis indicated that Uf-RTP1p and Us-RTP1p most likely enter the host cell as N-glycosylated proteins. However, the mechanism by which they cross the extrahaustorial membrane and accumulate in the host cytoplasm is unknown. The localization of RTP1p suggests that it might play an important role in the maintenance of the biotrophic interaction.

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