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

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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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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The 6th Amino Acid Mutation of Rep Protein Had No Effect on PCV2b but Enhanced PCV2d Virus Replication in Vitro

10.21203/rs.3.rs-202148/v1 ◽

2021 ◽

Author(s):

Xiaoyan Wu ◽

Shuo Wang ◽

Changxun Xin ◽

Chen Li ◽

Jianli Shi ◽

...

Keyword(s):

Amino Acid ◽

Viral Replication ◽

Porcine Circovirus Type ◽

Site Directed Mutagenesis ◽

Porcine Circovirus ◽

Wild Type ◽

Protein Amino Acid ◽

Rep Protein ◽

Significant Difference

Abstract Porcine circovirus type 2 (PCV2) is the etiological agent that primary cause of post-weaning multisystemic wasting syndrome (PMWS). The major genotypes, PCV2a, PCV2b and PCV2d, are highly prevalent, but now replaced with 2b and 2d in swine population in worldwide. Rep protein is the key protein for viral replication. Compared a large number of Rep protein amino acid (aa) sequences, we found that there were three sites with regular changes between 2b and 2d. In order to analyze the effect of key sites on viral replication, we used site-directed mutagenesis to mutate the 6th aa of Rep (alternations with asparagine and serine) between PCV2b and PCV2d, Two wild-type and two mutant viruses infectious clones were rescued by non-contaminated porcine kidney-15 (PK-15) cells. Real-time quantitative PCR and a one-step growth curve were used to determine viral load to assess the replication of rescued viruses. The results showed that there was no significant difference between the PCV2b mutation and the wild-type PCV2b virus in vitro, while the mutation ofPCV2d enhanced viral replication.

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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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Activation of the TLR/MyD88/NF-κB Signal Pathway Contributes to Changes in IL-4 and IL-12 Production in Piglet Lymphocytes Infected with Porcine Circovirus Type 2 In Vitro

PLoS ONE ◽

10.1371/journal.pone.0097653 ◽

2014 ◽

Vol 9 (5) ◽

pp. e97653 ◽

Cited By ~ 22

Author(s):

Dianning Duan ◽

Shuxia Zhang ◽

Xiaolin Li ◽

Hua Guo ◽

Mengmeng Chen ◽

...

Keyword(s):

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Signal Pathway ◽

Porcine Circovirus

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Two Amino Acid Mutations in the Capsid Protein of Type 2 Porcine Circovirus (PCV2) Enhanced PCV2 Replication In Vitro and Attenuated the Virus In Vivo

Journal of Virology ◽

10.1128/jvi.78.24.13440-13446.2004 ◽

2004 ◽

Vol 78 (24) ◽

pp. 13440-13446 ◽

Cited By ~ 78

Author(s):

M. Fenaux ◽

T. Opriessnig ◽

P. G. Halbur ◽

F. Elvinger ◽

X. J. Meng

Keyword(s):

Capsid Protein ◽

Vaccine Development ◽

Porcine Circovirus Type ◽

Postweaning Multisystemic Wasting Syndrome ◽

Porcine Circovirus ◽

Entire Genome ◽

Wasting Syndrome

ABSTRACT Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. To identify potential genetic determinants for virulence and replication, we serially passaged a PCV2 isolate 120 times in PK-15 cells. The viruses harvested at virus passages 1 (VP1) and 120 (VP120) were biologically, genetically, and experimentally characterized. The PCV2 VP120 virus replicated in PK-15 cells to a titer similar to that of the PK-15 cell line-derived nonpathogenic PCV1 but replicated more efficiently than PCV2 VP1 with a difference of about 1 log unit in the titers. The complete genomic sequences of viruses at passages 0, 30, 60, 90, and 120 were determined. After 120 passages, only two nucleotide mutations were identified in the entire genome, and both were located in the capsid gene: the mutations were located at nucleotide positions 328 (C328G) and 573 (A573C). The C328G mutation, in which a proline at position 110 of the capsid protein changed to an alanine (P110A), occurred at passage 30 and remained in the subsequent passages. The second mutation, A573C, resulting in a change from an arginine to a serine at position 191 (R191S), appeared at passage 120. To experimentally characterize the VP120 virus, 31 specific-pathogen-free pigs were randomly divided into three groups. Ten pigs in group 1 received phosphate-buffered saline as negative controls. Each pig in group 2 (11 pigs) was inoculated intramuscularly and intranasally with 104.9 50% tissue culture infective doses (TCID50) of PCV2 VP120. Each pig in group 3 (10 pigs) was similarly inoculated with 104.9 TCID50 of PCV2 VP1. Viremia was detected in 9 of 10 pigs in the PCV2 VP1 group with a mean duration of 3 weeks, but in only 4 of 11 pigs in the PCV2 VP120 group with a mean duration of 1.6 weeks. The PCV2 genomic copy numbers in serum in the PCV2 VP1 group were significantly higher than those in the PCV2 VP120 group (P < 0.0001). Gross and histopathologic lesions in pigs inoculated with PCV2 VP1 were more severe than those inoculated with PCV2 VP120 at both day 21 and 42 necropsies (P = 0.0032 and P = 0.0274, respectively). Taken together, the results from this study indicated that the P110A and R191S mutations in the capsid of PCV2 enhanced the growth ability of PCV2 in vitro and attenuated the virus in vivo. This finding has important implications for PCV2 vaccine development.

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