scholarly journals Betanodavirus B2 Is an RNA Interference Antagonist That Facilitates Intracellular Viral RNA Accumulation

2006 ◽  
Vol 80 (1) ◽  
pp. 85-94 ◽  
Author(s):  
Beau J. Fenner ◽  
Rekha Thiagarajan ◽  
Hui Kheng Chua ◽  
Jimmy Kwang
Keyword(s):  
Rna Interference ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Juvenile Fish ◽  
Nervous Necrosis Virus ◽  
Rna Transfection ◽  
B2 Rna ◽  
Rna Accumulation ◽  
High Level ◽  
Late Stages

ABSTRACT Betanodaviruses are small positive-sense bipartite RNA viruses that infect a wide variety of fish species and are notorious for causing lethal outbreaks in juvenile fish hatcheries worldwide. The function of a small nonstructural protein, B2, encoded by the subgenomic RNA3 of betanodaviruses, has remained obscure. Greasy grouper nervous necrosis virus, a betanodavirus model, was used to develop a facile DNA-based reverse genetics system that recapitulated the virus infection cycle, and we used this system to show that B2 is a small nonstructural protein that is essential for high level accumulation of viral RNA1 after RNA transfection of fish, mammalian, and avian cells. The defect in RNA1 accumulation in a B2 mutant was partially complemented by supplying B2 RNA in trans. Confocal analysis of the cellular distribution of B2 indicated that B2 is able to enter the nucleus and accumulates there during the late stages of GGNNV infection. Using human HeLa cells as a cellular RNA interference model, we found that B2 could efficiently antagonize RNA interference, which is a property shared by the distantly related alphanodavirus B2 proteins. This function provides appears to provide an explanation, at least in part, for why B2 mutant RNA1 is severely impaired in its intracellular accumulation.

scholarly journals Nodamura Virus Nonstructural Protein B2 Can Enhance Viral RNA Accumulation in both Mammalian and Insect Cells

2004 ◽  
Vol 78 (12) ◽  
pp. 6698-6704 ◽  
Author(s):  
Kyle L. Johnson ◽  
B. Duane Price ◽  
Lance D. Eckerle ◽  
L. Andrew Ball
Keyword(s):  
Rna Interference ◽  
Cell Lines ◽  
Nonstructural Protein ◽  
Viral Rna ◽  
Host Cells ◽  
Flock House Virus ◽  
Nonstructural Proteins ◽  
Reading Frame ◽  
Rna Accumulation ◽  
Nodamura Virus

ABSTRACT During infection of both vertebrate and invertebrate cell lines, the alphanodavirus Nodamura virus (NoV) expresses two nonstructural proteins of different lengths from the B2 open reading frame. The functions of these proteins have yet to be determined, but B2 of the related Flock House virus suppresses RNA interference both in Drosophila cells and in transgenic plants. To examine whether the NoV B2 proteins had similar functions, we compared the replication of wild-type NoV RNA with that of mutants unable to make the B2 proteins. We observed a defect in the accumulation of mutant viral RNA that varied in extent from negligible in some cell lines (e.g., baby hamster kidney cells) to severe in others (e.g., human HeLa and Drosophila DL-1 cells). These results are consistent with the notion that the NoV B2 proteins act to circumvent an innate antiviral response such as RNA interference that differs in efficacy among different host cells.

scholarly journals Identification of an intramolecular switch that controls the interaction of the helicase nsp10 with the membrane-associated nsp12 of porcine reproductive and respiratory syndrome virus

2021 ◽  
Author(s):  
Yunhao Hu ◽  
Purui Ke ◽  
Peng Gao ◽  
Yongning Zhang ◽  
Lei Zhou ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Interaction Network ◽  
Single Amino Acid ◽  
Respiratory Syndrome Virus ◽  
Subgenomic Rna ◽  
Linker Region ◽  
Transcription Complexes ◽  
Rna Accumulation

A critical step in replication of positive-stranded RNA viruses is the assembly of replication and transcription complexes (RTC). We have recently mapped the nonstructural protein (nsp) interaction network of porcine reproductive and respiratory syndrome virus (PRRSV) and provided evidence by truncation mutagenesis that the recruitment of viral core replicase enzymes (nsp9 and nsp10) to membrane proteins (nsp2, nsp3, nsp5 and nsp12) is subject to regulation. Here, we went further to discover an intramolecular switch within the helicase nsp10 that controls its interaction with the membrane-associated protein nsp12. Deletion of nsp10 linker region aa.124-133 connecting the domain 1B to 1A led to complete re-localization and co-localization in the cells co-expressing nsp12. Moreover, single amino acid substitutions (e.g., nsp10 E131A and I132A) were sufficient to enable the nsp10-nsp12 interaction. Further proof came from membrane floatation assays that revealed a clear movement of nsp10 mutants, but not WT nsp10, towards the top of sucrose gradients in the presence of nsp12. Interestingly, the same mutations were not able to activate the nsp10-nsp2/3 interaction, suggesting a differential requirement for conformation. Reverse genetics analysis showed that PRRSV mutants carrying the single substitutions were not viable and defective of subgenomic RNA (sg RNA) accumulation. Together, our results provide strong evidence for a regulated interaction between nsp10 and nsp12 and suggest an essential role for an orchestrated RTC assembly in sg RNA synthesis. IMPORTANCE Assembly of replication and transcription complexes (RTC) is a limiting step for viral RNA synthesis. The PRRSV RTC macromolecular complexes are comprised of mainly viral nonstructural replicase proteins (nsps), but how they come together remains elusive. We previously showed that viral helicase nsp10 interacts nsp12 in a regulated manner by truncation mutagenesis. Here we revealed that the interaction is controlled by single residues within the domain linker region of nsp10. Moreover, the activation mutations leads to defect in viral sg RNA synthesis. Our results provide important insight into the mechanisms of PRRSV RTC assembly and regulation of viral sg RNA synthesis.

scholarly journals The Porcine Reproductive and Respiratory Syndrome Virus nsp2 Cysteine Protease Domain Possesses both trans- and cis-Cleavage Activities

2009 ◽  
Vol 83 (18) ◽  
pp. 9449-9463 ◽  
Author(s):  
Jun Han ◽  
Mark S. Rutherford ◽  
Kay S. Faaberg
Keyword(s):  
Cysteine Protease ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Point Mutations ◽  
Respiratory Syndrome Virus ◽  
Protease Domain ◽  
Nonstructural Protein 2 ◽  
Cleavage Activity ◽  
Cysteine Protease Domain

ABSTRACT The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr47 to Cys240) and aa 47 to 323 (Tyr47 to Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55- His124 catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142, and Cys147). The conserved aspartic acids (e.g., Asp89) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197 dipeptide.

scholarly journals Mammalian Orthoreovirus Factories Modulate Stress Granule Protein Localization by Interaction with G3BP1

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Promisree Choudhury ◽  
Luke D. Bussiere ◽  
Cathy L. Miller
Keyword(s):  
Immune Response ◽  
Virus Infection ◽  
Innate Immune Response ◽  
Nonstructural Protein ◽  
Innate Immune ◽  
Common Mechanism ◽  
Effector Protein ◽  
Associated Proteins ◽  
Mammalian Orthoreovirus ◽  
Late Stages

ABSTRACT Mammalian orthoreovirus (MRV) infection induces phosphorylation of translation initiation factor eIF2α, which promotes the formation of discrete cytoplasmic inclusions, termed stress granules (SGs). SGs are emerging as a component of the innate immune response to virus infection, and modulation of SG assembly is a common mechanism employed by viruses to counter this antiviral response. We previously showed that MRV infection induces SGs early and then interferes with SG formation as infection proceeds. In this work, we found that SG-associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed virus factories (VFs), where viral transcription, translation, and replication occur. The localization of SG proteins to VFs was dependent on polysome dissociation and occurred via association of the SG effector protein, Ras-GAP SH3-binding protein 1 (G3BP1), with the MRV nonstructural protein σNS, which localizes to VFs via association with VF nucleating protein, μNS. Deletion analysis of the σNS RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed that σNS association and VF localization phenotypes of G3BP1 do not occur solely through RNA or ribosomal binding but require both the RRM and RGG domains of G3BP1 for maximal viral-factory-like structure (VFL) localization and σNS association. Coexpression of σNS and μNS resulted in disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a manner correlating with strain-dependent induction of host translation shutoff. These results suggest that σNS association with G3BP1 and relocalization of G3BP1 to the VF periphery play roles in SG disruption to facilitate MRV replication in the host translational shutoff environment. IMPORTANCE SGs and SG effector proteins have emerged as important, yet poorly understood, players in the host's innate immune response to virus infection. MRV infection induces SGs early during infection that are dispersed and/or prevented from forming during late stages of infection despite continued activation of the eIF2α signaling pathway. Cellular and viral components involved in disruption of SGs during late stages of MRV infection remain to be elucidated. This work provides evidence that MRV disruption of SGs may be facilitated by association of the MRV nonstructural protein σNS with the major SG effector protein G3BP1 and subsequent localization of G3BP1 and other SG-associated proteins around the peripheries of virus-encoded factories, interrupting the normal formation of SGs. Our findings also reveal the importance of G3BP1 as an inhibitor of MRV replication during infection for the first time.

scholarly journals Rearing larvae and juveniles of rainbow trout with different amino acid nutrition

2020 ◽  
Author(s):  
V. Kondratiuk ◽  
Keyword(s):  
Amino Acids ◽  
Rainbow Trout ◽  
Amino Acid ◽  
Juvenile Fish ◽  
Control Group ◽  
Economic Optimization ◽  
Larvae And Juveniles ◽  
Experimental Fish ◽  
Juvenile Trout ◽  
High Level

The article considers the efficiency of using complete compound feeds with different levels of lysine and methionine in rearing larvae and juveniles of rainbow trout. The purpose of the experiment was to establish the effect of different amino acid levels in nutrition of juvenile trout on its productivity. For this purpose, five experimental groups were formed by the method of analogues. The experiment lasted 35 days and was divided into two periods: equalizing (5 days) and main (30 days). During the equalizing period, the experimental fish consumed feed of the control group. In the main period, the levels of lysine and methionine in experimental feeds for different experimental groups of trout ranged from 2.9 to 3.3% and from 0.95 to 3.15%, respectively. It is proved that increasing the content of lysine and methionine in feed for larvae and juveniles of rainbow trout to the levels of 3.2 and 1.1%, respectively, promotes increase in their weight by 10.7% (p <0.01) and growth intensity - by 6.4-11.1%, while further increase in these amino acids content in the feed reduces the productivity of fish. Polynomial equations for determining the juvenile fish weight at any stage of rearing at a high level of determination for each of the experimental groups have been calculated. It was found that with an increase in the content of lysine and methionine in feed to the levels of 3.2 and 1.1%, respectively, reduces feed costs per 1 kg of weight gain in trout larvae and juveniles by 4.1%, and a further increase in levels of these amino acids to 3.3 and 1.15%, respectively, increase this index by 1.1%. The experimental fish survival ranged from 83.9 to 85.1%. In the production of trout according to the criteria of maximum productivity and to the economic optimization criteria, it is recommended to use complete feed with the level of lysine - 3.2% and methionine - 1.1% for feeding trout larvae and juveniles.

scholarly journals Population structure of an endemic fish Limia zonata (Cyprinodontiformes: Poeciliinae) in the Maimón River, Dominican Republic

2019 ◽  
pp. 22-38
Author(s):  
Patricia Torres-Pineda ◽  
Carlos M. Rodríguez-Peña ◽  
Óscar M. Lasso-Alcalá ◽  
Arlen Marmolejo
Keyword(s):  
Population Structure ◽  
Dominican Republic ◽  
Juvenile Fish ◽  
Fish Population ◽  
Size Classes ◽  
Relative Condition Factor ◽  
Good State ◽  
High Level ◽  
Rates Of Growth ◽  
The Relationship

 The population structure of Limia zonata (Nichols, 1915) was studied in four locations in the Maimón River, Monseñor Nouel province, Dominican Republic. The sex and adult-juveniles ratio was calculated for all sampled localities. It was found that the sex ratio of L. zonata was significantly different from 1: 1 and is biased towards females (1.0: 1.6). Juvenile fish were more abundant and frequent than adults (69.3%). More than 800 fish were measured and weighed to analyze the frequency of size classes, morphometry and body condition. Ten size classes were distinguished that vary in frequency according to sex. Total length of the specimens ranged from 6.50 to 65.69 mm, while weight varied between 0.004 and 3.941 g. The morphology varied between sexes and a pronounced sexual dimorphism was found. All the morphometric variables studied showed a high level of association (r). The adjustment of the regression models (r2) of the morphometric relationships was between 85% and 99%. The relationship between the standard length and the total weight was reflected in the rates of growth. The Relative Condition Factor (Kn) demonstrated the good state of health of this fish population, but also variations between the sexes were detected.

scholarly journals Growth-regulated expression of rhoG, a new member of the ras homolog gene family.

1992 ◽  
Vol 12 (7) ◽  
pp. 3138-3148 ◽  
Author(s):  
S Vincent ◽  
P Jeanteur ◽  
P Fort
Keyword(s):  
Gene Family ◽  
Transcriptional Activation ◽  
Small Gtpase ◽  
Lung Fibroblasts ◽  
Human Organs ◽  
Regulated Expression ◽  
Genes Encoding ◽  
Rna Stabilization ◽  
Rna Accumulation ◽  
High Level

Cellular transition from the resting state to DNA synthesis involves master switches genes encoding transcriptional factors (e.g., fos, jun, and egr genes), whose targets remain to be fully characterized. To isolate coding sequences specifically accumulated in late G1, a differential screening was performed on a cDNA library prepared from hamster lung fibroblasts stimulated for 5 h with serum. One of the positive clones which displayed a sevenfold induction, turned out to code for a protein sharing homology to Ras-like products. Cloning and sequence analysis of the human homolog revealed that this putative new small GTPase, referred to as rhoG, is more closely related to the rac, CDC42, and TC10 members of the rho (ras homolog) gene family and might have diverged very early during evolution. rhoG mRNA accumulates in proportion to the mitogenic strength of various purified growth factors used for the stimulation, as a consequence of transcriptional activation. G1-specific RNA accumulation is impaired upon addition of antimitogenic cyclic AMP and is enhanced when protein synthesis is inhibited, mainly as a result of RNA stabilization. rhoG mRNA expression is observed in a wide variety of human organs but reaches a particularly high level in lung and placental tissues.

scholarly journals Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

2015 ◽  
Vol 89 (9) ◽  
pp. 4907-4917 ◽  
Author(s):  
Anna M. Mielech ◽  
Xufang Deng ◽  
Yafang Chen ◽  
Eveline Kindler ◽  
Dorthea L. Wheeler ◽  
...  
Keyword(s):  
Viral Replication ◽  
Protease Activity ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Viral Pathogenesis ◽  
Wild Type ◽  
Viral Protease ◽  
Novel Approach ◽  
Protease Stability

ABSTRACTUbiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation.IMPORTANCEIntroducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

scholarly journals Optimizing RNA interference for application in mammalian cells

2004 ◽  
Vol 380 (3) ◽  
pp. 593-603 ◽  
Author(s):  
René H. MEDEMA
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Mutational Analysis ◽  
Life Sciences ◽  
Novel Technologies ◽  
Genome Wide ◽  
Technological Developments ◽  
Interfering Rna

Over the last 2 years, the scientific community has rapidly embraced novel technologies that allow gene silencing in vertebrates. Ease of application, cost effectiveness and the possibilities for genome-wide reverse genetics have quickly turned this approach into a widely accepted, almost mandatory asset for a self-respecting laboratory in life sciences. This review discusses some of the recent technological developments that allow the application of RNAi (RNA interference) in mammalian cells. In addition, the advantages of applying RNAi to study cell cycle events and the emerging approaches to perform mutational analysis by complementation in mammalian cells are evaluated. In addition, common pitfalls and drawbacks of RNAi will be reviewed, as well as the possible ways to get around these shortcomings of gene silencing by small interfering RNA.

scholarly journals The chicken c-erbA proto-oncogene is preferentially expressed in erythrocytic cells during late stages of differentiation.

1987 ◽  
Vol 7 (7) ◽  
pp. 2416-2424 ◽  
Author(s):  
D Hentzen ◽  
A Renucci ◽  
D le Guellec ◽  
M Benchaibi ◽  
P Jurdic ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Life Span ◽  
Blood Cells ◽  
Low Levels ◽  
High Level ◽  
Nuclease Mapping ◽  
Kidney Liver ◽  
Late Stages ◽  
Different Tissues

We analyzed the expression of the c-erbA proto-oncogene in different tissues of chicken embryos. c-erbA transcripts were found at low levels in the lung, kidney, liver, and heart and in high amounts in embryonic blood cells. Nuclease mapping assays proved that these transcripts were true c-erbA transcripts. In situ hybridization on fractionated embryonic blood cells showed that c-erbA transcripts were predominantly found in erythroblasts, particularly during the final step of differentiation. Life span analysis of c-erbA mRNAs revealed their relative instability, demonstrating that the high level of c-erbA transcripts in embryonic erythroblasts was not the result of passive accumulation. These results suggest that the c-erbA genes play some role in erythrocyte differentiation.

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