An Ascovirus-Encoded RNase III Autoregulates Its Expression and Suppresses RNA Interference-Mediated Gene Silencing

ABSTRACT RNase III proteins play vital roles in processing of several types of RNA molecules and gene silencing. Recently, it has been discovered that some plant and animal viruses encode RNase III-like proteins as well. Genome sequencing of four virus species belonging to the Ascoviridae family has revealed sequence conservation of an RNase III open reading frame among the viruses. These have not been explored in ascoviruses, and therefore their role in host-virus interaction is unknown. Here, we confirmed expression of Heliothis virescens ascovirus (HvAV-3e) open reading frame 27 (orf27) that encodes an RNase III-like protein after infection and demonstrated dsRNA specific endoribonuclease activity of the encoded protein. Analysis of the expression patterns of orf27 in virus-infected insect cells and a bacterial expression system revealed autoregulation of this protein over time. Moreover, HvAV-3e RNase III was found essential for virus DNA replication and infection using RNA interference (RNAi)-mediated gene silencing. In addition, using green fluorescent protein gene as a marker, we provide evidence that RNase III is involved in the suppression of gene silencing. To our knowledge, this is the first insect virus-encoded RNase III described and shown to suppress host cell RNAi defense mechanism.

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An Alternative Platform for Protein Expression Using an Innate Whole Expression Module from Metagenomic DNA

Microorganisms ◽

10.3390/microorganisms7010009 ◽

2019 ◽

Vol 7 (1) ◽

pp. 9 ◽

Cited By ~ 2

Author(s):

Dae-Eun Cheong ◽

So-Youn Park ◽

Ho-Dong Lim ◽

Geun-Joong Kim

Keyword(s):

Gene Expression ◽

Regulatory Element ◽

Expression System ◽

Expression Patterns ◽

Gene Clusters ◽

Single Element ◽

Reading Frame ◽

Cis Acting ◽

Dna Libraries ◽

Expression Module

Many integrated gene clusters beyond a single genetic element are commonly trapped as the result of promoter traps in (meta)genomic DNA libraries. Generally, a single element, which is mainly the promoter, is deduced from the resulting gene clusters and employed to construct a new expression vector. However, expression patterns of target proteins under the incorporated promoter are often inconsistent with those shown in clones harboring plasmids with gene clusters. These results suggest that the integrated set of gene clusters with diverse cis- and trans-acting elements is evolutionarily tuned as a complete set for gene expression, and is an expression module with all the components for the expression of a nested open reading frame (ORF). This possibility is further supported by truncation and/or serial deletion analysis of this module in which the expression of the nested ORF is highly fluctuated or reduced frequently, despite being supported by plentiful cis-acting elements in the spanning regions around the ORF such as the promoter, ribosome binding site (RBS), terminator, and 3′-/5′-UTRs for gene expression. Here, we examined whether an innate module with a naturally overexpressed gene could be considered as a scaffold for an expression system. For a proof-of-principle study, we mined a complete expression module with an innately overexpressed ORF in E. coli from a metagenomics DNA library, and incorporated it into a vector that had no regulatory element for expressing the insert. We obtained successful expression of several inserts such as MBP, GFPuv, β-glucosidase, and esterase using this simple construct without tuning and codon optimization of the target insert.

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Evidence That RNA Silencing-Mediated Resistance to Beet necrotic yellow vein virus Is Less Effective in Roots Than in Leaves

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0194 ◽

2005 ◽

Vol 18 (3) ◽

pp. 194-204 ◽

Cited By ~ 71

Author(s):

Ida Bagus Andika ◽

Hideki Kondo ◽

Tetsuo Tamada

Keyword(s):

Nicotiana Benthamiana ◽

Rna Silencing ◽

Defense Mechanism ◽

Virus Titer ◽

Virus Multiplication ◽

Open Reading Frame ◽

Yellow Vein ◽

Polymyxa Betae ◽

Small Interfering Rnas ◽

Reading Frame

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.

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Cryphonectria hypovirus 3, a Virus Species in the Family Hypoviridae with a Single Open Reading Frame

Virology ◽

10.1006/viro.1999.0039 ◽

1999 ◽

Vol 265 (1) ◽

pp. 66-73 ◽

Cited By ~ 64

Author(s):

C.D. Smart ◽

W. Yuan ◽

R. Foglia ◽

D.L. Nuss ◽

D.W. Fulbright ◽

...

Keyword(s):

Open Reading Frame ◽

Single Open Reading Frame ◽

Virus Species ◽

Reading Frame ◽

The Family ◽

Cryphonectria Hypovirus

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A Streptomyces coelicolor Antibiotic Regulatory Gene, absB, Encodes an RNase III Homolog

Journal of Bacteriology ◽

10.1128/jb.181.19.6142-6151.1999 ◽

1999 ◽

Vol 181 (19) ◽

pp. 6142-6151 ◽

Cited By ~ 36

Author(s):

Brenda Price ◽

Trifon Adamidis ◽

Renqui Kong ◽

Wendy Champness

Keyword(s):

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Regulatory Gene ◽

Open Reading Frame ◽

Dependent Manner ◽

Rnase Iii ◽

Reading Frame ◽

E Coli ◽

Lines Of Evidence ◽

Global Defect

ABSTRACT Streptomyces coelicolor produces four genetically and structurally distinct antibiotics in a growth-phase-dependent manner.S. coelicolor mutants globally deficient in antibiotic production (Abs− phenotype) have previously been isolated, and some of these were found to define the absB locus. In this study, we isolated absB-complementing DNA and show that it encodes the S. coelicolor homolog of RNase III (rnc). Several lines of evidence indicate that theabsB mutant global defect in antibiotic synthesis is due to a deficiency in RNase III. In marker exchange experiments, the S. coelicolor rnc gene rescued absB mutants, restoring antibiotic production. Sequencing the DNA of absB mutants confirmed that the absB mutations lay in thernc open reading frame. Constructed disruptions ofrnc in both S. coelicolor 1501 andStreptomyces lividans 1326 caused an Abs−phenotype. An absB mutation caused accumulation of 30S rRNA precursors, as had previously been reported for E. coli rncmutants. The absB gene is widely conserved in streptomycetes. We speculate on why an RNase III deficiency could globally affect the synthesis of antibiotics.

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Identification and Characterization of alcR, a Gene Encoding an AraC-Like Regulator of Alcaligin Siderophore Biosynthesis and Transport in Bordetella pertussis andBordetella bronchiseptica

Journal of Bacteriology ◽

10.1128/jb.180.4.862-870.1998 ◽

1998 ◽

Vol 180 (4) ◽

pp. 862-870 ◽

Cited By ~ 39

Author(s):

Fiona C. Beaumont ◽

Ho Young Kang ◽

Timothy J. Brickman ◽

Sandra K. Armstrong

Keyword(s):

Bordetella Pertussis ◽

Transcription Initiation ◽

Expression System ◽

Protein A ◽

Initiation Site ◽

Open Reading Frame ◽

Growth Defect ◽

Nucleotide Sequencing ◽

Reading Frame ◽

Siderophore Biosynthesis

ABSTRACT A Bordetella bronchiseptica iron transport mutant was isolated following an enrichment procedure based on streptonigrin resistance. The mutant displayed a growth defect on iron-restricted medium containing ferric alcaligin as the sole iron source. In addition to the apparent inability to acquire iron from the siderophore, the mutant failed to produce alcaligin as well as two known iron-regulated proteins, one of which is the AlcC alcaligin biosynthesis protein. A 1.6-kb KpnI-PstI Bordetella pertussis DNA fragment mapping downstream of the alcaligin biosynthesis genes alcABC restored both siderophore biosynthesis and expression of the iron-regulated proteins to the mutant. Nucleotide sequencing of this complementing 1.6-kb region identified an open reading frame predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose the gene designation alcR. Primer extension analysis localized an iron-regulated transcription initiation site upstream of the alcR open reading frame and adjacent to sequences homologous to the consensus Fur repressor binding site. The AlcR protein was produced by using an Escherichia coli expression system and visualized in electrophoretic gels. In-frame alcR deletion mutants of B. pertussisand B. bronchiseptica were constructed, and the defined mutants exhibited the alcR mutant phenotype, characterized by the inability to produce and transport alcaligin and express the two iron-repressed proteins. The cloned alcR gene provided intrans restored these siderophore system activities to the mutants. Together, these results indicate that AlcR is involved in the regulation of Bordetella alcaligin biosynthesis and transport genes and is required for their full expression.

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SufS Is a NifS-Like Protein, and SufD Is Necessary for Stability of the [2Fe-2S] FhuF Protein in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.181.10.3307-3309.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3307-3309 ◽

Cited By ~ 104

Author(s):

Silke I. Patzer ◽

Klaus Hantke

Keyword(s):

Escherichia Coli ◽

Type Strain ◽

Wild Type Strain ◽

Expression System ◽

Open Reading Frame ◽

Wild Type ◽

Reading Frame ◽

His Tag ◽

The Family ◽

Ferrioxamine B

ABSTRACT Escherichia coli fhuF mutants, asufS::MudI mutant, and asufD::MudI mutant were found to have the same phenotype: the inability to use ferrioxamine B as an iron source in a plate assay. In addition, the sufS and sufDgenes were shown to be regulated by the iron-dependent Fur repressor. Sequence analysis revealed that the sufS open reading frame corresponds to orf f406. The protein SufS belongs to the family of NifS-like proteins, which supply sulfur for [Fe-S] centers. The protein FhuF contains a [2Fe-2S] center. A mutation in the upstream sufD gene (orf f423) caused the same phenotype. The T7 expression system and a His tag allow the isolation in good yield of the FhuF protein from a wild-type strain. In contrast, overproduction of the protein in a ΔsufD strain failed. Radioactive labeling of N-His-FhuF with [35S]methionine showed that the protein was unstable in the ΔsufD mutant.

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Differences in the expression of the hepatitis C virus core+1 open reading frame between a nuclear and a cytoplasmic expression system

Journal of General Virology ◽

10.1099/vir.0.83260-0 ◽

2008 ◽

Vol 89 (1) ◽

pp. 222-231 ◽

Cited By ~ 10

Author(s):

Niki Vassilaki ◽

Katerina I. Kalliampakou ◽

Penelope Mavromara

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Expression System ◽

Open Reading Frame ◽

Reading Frame ◽

Transcription System ◽

The Core ◽

Internal Core

The hepatitis C virus (HCV) genome possesses an open reading frame (ORF) overlapping the core gene at +1 nucleotide (core+1 ORF). Initial in vitro studies suggested that the core+1 ORF is translated by a ribosomal −2/+1 frameshift mechanism during elongation of the viral polyprotein. Recent studies, however, based on transfection of mammalian cells with reporter constructs have shown that translation of the core+1 ORF is mediated from internal core+1 codons. To resolve the apparent discrepancies associated with the mechanism of core+1 translation, we examined the expression of the HCV-1 and HCV-1a (H) core+1 ORF in a cytoplasmic transcription system based on Huh-7/T7 cells that constitutively synthesize the T7 RNA polymerase in comparison to that in Huh-7 cells. We showed that the efficiency of both the −2/+1 and −1/+2 frameshift events operating at the HCV-1 core codons 8–11 is significantly enhanced in the Huh-7/T7 cytoplasmic transcription system and is dependent on the presence of the consecutive adenine (A) residues within core codons 8–11. In contrast, internal translation initiation at core+1 codons 85/87 occurs in both the nuclear and cytoplasmic transcription systems and is not repressed by the ribosomal frameshifting event. Finally, although core+1 codons 85/87 is the most efficient site for internal initiation of core+1 translation, it may not be unique, as additional internal core+1 codon(s) appear to drive translation at low levels.

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Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Open Reading Frame 4 Protein (Kaposica) Is a Functional Homolog of Complement Control Proteins

Journal of Virology ◽

10.1128/jvi.77.6.3878-3881.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3878-3881 ◽

Cited By ~ 41

Author(s):

Jayati Mullick ◽

John Bernet ◽

Akhilesh K. Singh ◽

John D. Lambris ◽

Arvind Sahu

Keyword(s):

Kaposi's Sarcoma ◽

Expression System ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Open Reading Frame ◽

Reading Frame ◽

Complement Control Proteins ◽

Functional Homolog ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The genome analysis of Kaposi's sarcoma-associated herpesvirus (KSHV) has revealed the presence of an open reading frame (ORF 4) with sequence homology to complement control proteins. To assign a function to this protein, we have now expressed this ORF using the Pichia expression system and shown that the purified protein inhibited human complement-mediated lysis of erythrocytes, blocked cell surface deposition of C3b (the proteolytically activated form of C3), and served as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b (the subunits of C3 convertases). Thus, our data indicate that this KSHV inhibitor of complement activation (kaposica) provides a mechanism by which KSHV can subvert complement attack by the host.

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Inhibition of Gammaherpesvirus Replication by RNA Interference

Journal of Virology ◽

10.1128/jvi.77.5.3301-3306.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3301-3306 ◽

Cited By ~ 71

Author(s):

Qingmei Jia ◽

Ren Sun

Keyword(s):

Rna Interference ◽

Viral Gene Expression ◽

Open Reading Frame ◽

Viral Gene ◽

Specific Gene ◽

Small Interfering Rnas ◽

Viral Production ◽

Reading Frame ◽

Early Protein ◽

Expression Program

ABSTRACT RNA interference (RNAi) is a conserved mechanism in which double-stranded, small interfering RNAs (siRNAs) trigger a sequence-specific gene-silencing process. Here we describe the inhibition of murine herpesvirus 68 replication by siRNAs targeted to sequences encoding Rta, an immediate-early protein known as an initiator of the lytic viral gene expression program, and open reading frame 45 (ORF 45), a conserved viral protein. Our results suggest that RNAi can block gammaherpesvirus replication and ORF 45 is required for efficient viral production.

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Divergent Antiviral Mechanisms of Two Viperin Homeologs in a Recurrent Polyploid Fish

Frontiers in Immunology ◽

10.3389/fimmu.2021.702971 ◽

2021 ◽

Vol 12 ◽

Author(s):

Cheng-Yan Mou ◽

Shun Li ◽

Long-Feng Lu ◽

Yang Wang ◽

Peng Yu ◽

...

Keyword(s):

Crucian Carp ◽

Expression Patterns ◽

Protein A ◽

Open Reading Frame ◽

Gibel Carp ◽

Duplicated Genes ◽

Reading Frame ◽

Screening Analysis ◽

Α Helix ◽

Shed Light

Polyploidy and subsequent diploidization provide genomic opportunities for evolutionary innovations and adaptation. The researches on duplicated gene evolutionary fates in recurrent polyploids have seriously lagged behind that in paleopolyploids with diploidized genomes. Moreover, the antiviral mechanisms of Viperin remain largely unclear in fish. Here, we elaborate the distinct antiviral mechanisms of two viperin homeologs (Cgviperin-A and Cgviperin-B) in auto-allo-hexaploid gibel carp (Carassius gibelio). First, Cgviperin-A and Cgviperin-B showed differential and biased expression patterns in gibel carp adult tissues. Subsequently, using co-immunoprecipitation (Co-IP) screening analysis, both CgViperin-A and CgViperin-B were found to interact with crucian carp (C. auratus) herpesvirus (CaHV) open reading frame 46 right (ORF46R) protein, a negative herpesvirus regulator of host interferon (IFN) production, and to promote the proteasomal degradation of ORF46R via decreasing K63-linked ubiquitination. Additionally, CgViperin-B also mediated ORF46R degradation through autophagosome pathway, which was absent in CgViperin-A. Moreover, we found that the N-terminal α-helix domain was necessary for the localization of CgViperin-A and CgViperin-B at the endoplasmic reticulum (ER), and the C-terminal domain of CgViperin-A and CgViperin-B was indispensable for the interaction with degradation of ORF46R. Therefore, the current findings clarify the divergent antiviral mechanisms of the duplicated viperin homeologs in a recurrent polyploid fish, which will shed light on the evolution of teleost duplicated genes.

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