Analysis of transcription of Porcine circovirus type 1

Porcine circovirus type 1 (PCV1) contains two major open reading frames encoding the replication initiator proteins, Rep and Rep′, and the structural protein, Cap. The promoters of these two genes (P cap and P rep ) have been mapped. P cap is located within the rep open reading frame (nt 1328–1252). P rep has been mapped to the intergenic region immediately upstream of the rep gene (nt 640–796) and overlaps the origin of replication of PCV1. Although binding of both rep gene products to a fragment containing P rep and the overlapping origin of replication has been reported, only the full-length Rep protein repressed P rep , while the spliced isoform Rep′ did not. P rep repression is mediated by binding of the Rep protein to the two inner hexamers, H1 and H2, located in the origin of PCV1, whereas binding of Rep to hexamers H3 and H4 was not necessary. Use of Rep mutants indicated that the conserved rolling-circle replication domain II as well as the P loop are essential for repression of P rep . In contrast to P rep , transcription of P cap was not influenced by viral proteins. Additionally, the ratio of the rep and rep′ transcripts was analysed. Twelve hours after transfection of PK15 cells with an infectious clone of PCV1, similar amounts of both transcripts were detected, but later the amount of the two transcripts varied, indicating a balanced expression of the two rep transcripts.

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Rep and Rep′ Protein of Porcine circovirus Type 1 Bind to the Origin of Replication in Vitro

Virology ◽

10.1006/viro.2001.1203 ◽

2001 ◽

Vol 291 (1) ◽

pp. 152-160 ◽

Cited By ~ 56

Author(s):

Tobias Steinfeldt ◽

Tim Finsterbusch ◽

Annette Mankertz

Keyword(s):

Porcine Circovirus Type ◽

Porcine Circovirus ◽

Origin Of Replication ◽

Rep Protein

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Functional Analysis of cis- and trans-Acting Replication Factors of Porcine Circovirus Type 1

Journal of Virology ◽

10.1128/jvi.02420-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5696-5704 ◽

Cited By ~ 22

Author(s):

Tobias Steinfeldt ◽

Tim Finsterbusch ◽

Annette Mankertz

Keyword(s):

Viral Replication ◽

Porcine Circovirus Type ◽

Porcine Circovirus ◽

Rolling Circle Replication ◽

Viral Origin ◽

Stem Loop ◽

Rolling Circle ◽

Rep Proteins

ABSTRACT The replication proteins Rep and Rep′ of porcine circovirus type 1 (PCV1) are both capable of introducing and resealing strand discontinuities at the viral origin of DNA replication in vitro underlying genome amplification by rolling-circle replication. The PCV1 origin of replication encompasses the minimal binding site (MBS) of the Rep and Rep′ proteins and an inverted repeat with the potential to form a stem-loop. In this study, both elements of the PCV1 origin were demonstrated to be essential for viral replication in transfected cells. Furthermore, investigation of conserved amino acid motifs within Rep and Rep′ proteins revealed that the mutation of motifs I, II, and III and of the GKS box interfered with viral replication. In vitro studies demonstrated that motifs I to III were essential for origin cleavage, while the GKS box was dispensable for the initiation of viral replication. A covalent link between Rep/Rep′ and the DNA after origin cleavage was demonstrated, providing a mechanism for energy conservation for the termination of replication.

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Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein

Journal of General Virology ◽

10.1099/0022-1317-81-9-2281 ◽

2000 ◽

Vol 81 (9) ◽

pp. 2281-2287 ◽

Cited By ~ 320

Author(s):

Porntippa Nawagitgul ◽

Igor Morozov ◽

Steven R. Bolin ◽

Perry A. Harms ◽

Steven D. Sorden ◽

...

Keyword(s):

Molecular Mass ◽

Gene Product ◽

Protein S ◽

Basic Amino Acid ◽

Porcine Circovirus Type ◽

Open Reading Frames ◽

Porcine Circovirus ◽

Structural Protein ◽

Major Structural Protein ◽

Orf2 Protein

Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa.

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Detection of Template Strand Switching during Initiation and Termination of DNA Replication of Porcine Circovirus

Journal of Virology ◽

10.1128/jvi.78.8.4268-4277.2004 ◽

2004 ◽

Vol 78 (8) ◽

pp. 4268-4277 ◽

Cited By ~ 26

Author(s):

Andrew K. Cheung

Keyword(s):

Dna Replication ◽

Porcine Circovirus Type ◽

Porcine Circovirus ◽

Inverted Repeats ◽

Minus Strand ◽

Gene Correction ◽

Melting Pot ◽

Rolling Circle ◽

Rep Protein ◽

Template Strand

ABSTRACT Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle “melting-pot” model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a “melted” configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a “gene correction” or “terminal repeat correction” mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.

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Detection and analysis of porcine circovirus type 1 in Hungarian wild boars: Short communication

Acta Veterinaria Hungarica ◽

10.1556/avet.56.2008.1.15 ◽

2008 ◽

Vol 56 (1) ◽

pp. 139-144 ◽

Cited By ~ 9

Author(s):

Attila Cságola ◽

István Kiss ◽

Tamás Tuboly

Keyword(s):

Porcine Circovirus Type ◽

Porcine Circovirus ◽

Wild Boars ◽

Chain Reactions ◽

Polymerase Chain Reactions ◽

Pathogenic Virus ◽

A Genome ◽

Domestic Swine ◽

Porcine Origin

Porcine circovirus type 1 (PCV1) is considered to be a non-pathogenic virus detected in cell cultures, vaccines or products used for cell culture preparations, all of them of porcine origin. Serological evidence and genetic studies suggested that PCV1 was widespread in domestic pigs. The presence of PCV1 in wild boars in Germany was also described using serological methods. This paper reports the first detection of PCV1 in Hungarian wild boars. Samples were collected at slaughterhouses and processed for polymerase chain reactions. The complete genome of PCV1 detected in the samples was determined and compared with the available PCV1 sequences of the GenBank database. The genomes formed two distinct clusters with minimum differences, where the Hungarian wild boar PCV1 (WB-H8) grouped together with genomes originating from domestic swine from China and Australia and with a genome detected in a porcine pepsin product.

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Attenuation of porcine circovirus type-2b by replacement with the Rep gene of porcine circovirus type-1

Virus Research ◽

10.1016/j.virusres.2013.02.007 ◽

2013 ◽

Vol 173 (2) ◽

pp. 270-279 ◽

Cited By ~ 4

Author(s):

Tao Hua ◽

Xianwei Wang ◽

Juan Bai ◽

Lili Zhang ◽

Jie Liu ◽

...

Keyword(s):

Porcine Circovirus Type ◽

Porcine Circovirus

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Porcine circovirus type 1 was undetected in vaccine but could be cultured in the cell substrate of Lanzhou lamb rotavirus vaccine

Journal of General Virology ◽

10.1099/jgv.0.000875 ◽

2018 ◽

Vol 99 (1) ◽

pp. 103-108

Author(s):

Qingchuan Yu ◽

Yan Liu ◽

Jialiang Du ◽

Yueyue Liu ◽

Lili Zhang ◽

...

Keyword(s):

Porcine Circovirus Type ◽

Porcine Circovirus ◽

Rotavirus Vaccine ◽

Cell Substrate

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Molecular Analysis of pDL10 from Acidianus ambivalens Reveals a Family of Related Plasmids from Extremely Thermophilic and Acidophilic Archaea

Genetics ◽

10.1093/genetics/152.4.1307 ◽

1999 ◽

Vol 152 (4) ◽

pp. 1307-1314

Author(s):

Arnulf Kletzin ◽

Angelika Lieke ◽

Tim Urich ◽

Robert L Charlebois ◽

Christoph W Sensen

Keyword(s):

Amino Acid ◽

Regulatory Protein ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Mung Bean Nuclease ◽

Stem Loop ◽

Rolling Circle ◽

Rep Protein ◽

Homologous Protein ◽

Rep Proteins

Abstract The 7598-bp plasmid pDL10 from the extremely thermophilic, acidophilic, and chemolithoautotrophic Archaeon Acidianus ambivalens was sequenced. It contains 10 open reading frames (ORFs) organized in five putative operons. The deduced amino acid sequence of the largest ORF (909 aa) showed similarity to bacterial Rep proteins known from phages and plasmids with rolling-circle (RC) replication. From the comparison of the amino acid sequences, a novel family of RC Rep proteins was defined. The pDL10 Rep protein shared 45-80% identical residues with homologous protein genes encoded by the Sulfolobus islandicus plasmids pRN1 and pRN2. Two DNA regions capable of forming extended stem-loop structures were also conserved in the three plasmids (48-69% sequence identity). In addition, a putative plasmid regulatory protein gene (plrA) was found, which was conserved among the three plasmids and the conjugative Sulfolobus plasmid pNOB8. A homolog of this gene was also found in the chromosome of S. solfataricus. Single-stranded DNA of both pDL10 strands was detected with a mung bean nuclease protection assay using PCR detection of protected fragments, giving additional evidence for an RC mechanism of replication.

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Autonomous Replication of the Conjugative Transposon Tn916

Journal of Bacteriology ◽

10.1128/jb.00639-16 ◽

2016 ◽

Vol 198 (24) ◽

pp. 3355-3366 ◽

Cited By ~ 21

Author(s):

Laurel D. Wright ◽

Alan D. Grossman

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Origin Of Replication ◽

Rolling Circle Replication ◽

Conjugative Transposon ◽

Rolling Circle ◽

Autonomous Replication ◽

Integrative And Conjugative Elements ◽

Rolling Circle Mechanism ◽

Origin Of Transfer

ABSTRACTIntegrative and conjugative elements (ICEs), also known as conjugative transposons, are self-transferable elements that are widely distributed among bacterial phyla and are important drivers of horizontal gene transfer. Many ICEs carry genes that confer antibiotic resistances to their host cells and are involved in the dissemination of these resistance genes. ICEs reside in host chromosomes but under certain conditions can excise to form a plasmid that is typically the substrate for transfer. A few ICEs are known to undergo autonomous replication following activation. However, it is not clear if autonomous replication is a general property of many ICEs. We found that Tn916, the first conjugative transposon identified, replicates autonomously via a rolling-circle mechanism. Replication of Tn916was dependent on the relaxase encoded byorf20of Tn916. The origin of transfer of Tn916,oriT(916), also functioned as an origin of replication. Using immunoprecipitation and mass spectrometry, we found that the relaxase (Orf20) and the two putative helicase processivity factors (Orf22 and Orf23) encoded by Tn916likely interact in a complex and that the Tn916relaxase contains a previously unidentified conserved helix-turn-helix domain in its N-terminal region that is required for relaxase function and replication. Lastly, we identified a functional single-strand origin of replication (sso) in Tn916that we predict primes second-strand synthesis during rolling-circle replication. Together these results add to the emerging data that show that several ICEs replicate via a conserved, rolling-circle mechanism.IMPORTANCEIntegrative and conjugative elements (ICEs) drive horizontal gene transfer and the spread of antibiotic resistances in bacteria. ICEs reside integrated in a host genome but can excise to create a plasmid that is the substrate for transfer to other cells. Here we show that Tn916, an ICE with broad host range, undergoes autonomous rolling-circle replication when in the plasmid form. We found that the origin of transfer functions as a double-stranded origin of replication and identified a single-stranded origin of replication. It was long thought that ICEs do not undergo autonomous replication. Our work adds to the evidence that ICEs replicate autonomously as part of their normal life cycle and indicates that diverse ICEs use the same replicative mechanism.

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