A Single Rep Protein Initiates Replication of Multiple Genome Components of Faba Bean Necrotic Yellows Virus, a Single-Stranded DNA Virus of Plants

ABSTRACT Faba bean necrotic yellows virus (FBNYV) belongs to the nanoviruses, plant viruses whose genome consists of multiple circular single-stranded DNA components. Eleven distinct DNAs, 5 of which encode different replication initiator (Rep) proteins, have been identified in two FBNYV isolates. Origin-specific DNA cleavage and nucleotidyl transfer activities were shown for Rep1 and Rep2 proteins in vitro, and their essential tyrosine residues that catalyze these reactions were identified by site-directed mutagenesis. In addition, we showed that Rep1 and Rep2 proteins hydrolyze ATP, and by changing the key lysine residue in the proteins’ nucleoside triphosphate binding sites, demonstrated that this ATPase activity is essential for multiplication of virus DNA in vivo. Each of the five FBNYV Rep proteins initiated replication of the DNA molecule by which it was encoded, but only Rep2 was able to initiate replication of all the six other genome components. Furthermore, of the fiverep components, only the Rep2-encoding DNA was always detected in 55 FBNYV samples from eight countries. These data provide experimental evidence for a master replication protein encoded by a multicomponent single-stranded DNA virus.

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An Adeno-Associated Virus (AAV) Initiator Protein, Rep78, Catalyzes the Cleavage and Ligation of Single-Stranded AAV ori DNA

Journal of Virology ◽

10.1128/jvi.74.7.3122-3129.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3122-3129 ◽

Cited By ~ 46

Author(s):

Richard H. Smith ◽

Robert M. Kotin

Keyword(s):

Dna Cleavage ◽

Site Directed Mutagenesis ◽

Nucleoside Triphosphate ◽

Sequence Motifs ◽

Rolling Circle Replication ◽

Mobility Shift ◽

Single Stranded Dna ◽

Adeno Associated Virus ◽

Rolling Circle ◽

Dna Substrates

ABSTRACT The Rep78 protein of adeno-associated virus (AAV) contains amino acid sequence motifs common to rolling-circle replication (RCR) initiator proteins. In this report, we describe RCR initiator-like activities of Rep78. We demonstrate that a maltose-binding protein (MBP)–Rep78 fusion protein can catalyze the cleavage and ligation of single-stranded DNA substrates derived from the AAV origin of replication. Rep-mediated single-stranded DNA cleavage was strictly dependent on the presence of certain divalent cations (e.g., Mn2+ or Mg2+) but did not require the presence of a nucleoside triphosphate cofactor. Electrophoretic mobility shift assays demonstrated that binding of single-stranded DNA by MBP-Rep78 was influenced by the length of the substrate as well as the presence of potential single-stranded cis-acting sequence elements. Site-directed mutagenesis was used to examine the role of specific tyrosine residues within a conserved RCR motif (motif 3) of Rep78. Replacement of Tyr-156 with phenylalanine abolished the ability of MBP-Rep78 to mediate the cleavage and ligation of single-stranded DNA substrates but not the ability to stably bind single-stranded DNA. The cleaving-joining activity of Rep78 is consistent with the mechanism of replicative intermediate dimer resolution proposed for the autonomous parvoviruses and may have implications for targeted integration of recombinant AAV vectors.

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Adeno-Associated Virus (AAV) Type 5 Rep Protein Cleaves a Unique Terminal Resolution Site Compared with Other AAV Serotypes

Journal of Virology ◽

10.1128/jvi.73.5.4293-4298.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4293-4298 ◽

Cited By ~ 51

Author(s):

John A. Chiorini ◽

Sandra Afione ◽

Robert M. Kotin

Keyword(s):

Dna Sequences ◽

Cleavage Site ◽

Inverted Terminal Repeat ◽

Nonstructural Proteins ◽

Adeno Associated Virus ◽

Rep Protein ◽

Rep Proteins ◽

A Genome ◽

Viral Components

ABSTRACT Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) intrans, and inverted terminal repeat (ITR) sequences incis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.

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Novel circular DNA virus identified in Opuntia discolor (Cactaceae) that codes for proteins with similarity to those of geminiviruses

Journal of General Virology ◽

10.1099/jgv.0.001671 ◽

2021 ◽

Vol 102 (11) ◽

Author(s):

Rafaela S. Fontenele ◽

Matias Köhler ◽

Lucas C. Majure ◽

Jesús A. Avalos-Calleros ◽

Gerardo R. Argüello-Astorga ◽

...

Keyword(s):

South America ◽

Rolling Circle Replication ◽

Dna Virus ◽

Single Stranded Dna ◽

Dna Viruses ◽

Circular Dna ◽

Rolling Circle ◽

Rep Protein ◽

Molecular Features ◽

Viral Components

Viral metagenomic studies have enabled the discovery of many unknown viruses and revealed that viral communities are much more diverse and ubiquitous than previously thought. Some viruses have multiple genome components that are encapsidated either in separate virions (multipartite viruses) or in the same virion (segmented viruses). In this study, we identify what is possibly a novel bipartite plant-associated circular single-stranded DNA virus in a wild prickly pear cactus, Opuntia discolor, that is endemic to the Chaco ecoregion in South America. Two ~1.8 kb virus-like circular DNA components were recovered, one encoding a replication-associated protein (Rep) and the other a capsid protein (CP). Both of the inferred protein sequences of the Rep and CP are homologous to those encoded by members of the family Geminiviridae. These two putatively cognate components each have a nonanucleotide sequence within a likely hairpin structure that is homologous to the origins of rolling-circle replication (RCR), found in diverse circular single-stranded DNA viruses. In addition, the two components share similar putative replication-associated iterative sequences (iterons), which in circular single-stranded DNA viruses are important for Rep binding during the initiation of RCR. Such molecular features provide support for the possible bipartite nature of this virus, which we named utkilio virus (common name of the Opuntia discolor in South America) components A and B. In the infectivity assays conducted in Nicotiana benthamiana plants, only the A component of utkilio virus, which encodes the Rep protein, was found to move and replicate systemically in N. benthamiana. This was not true for component B, for which we did not detect replication, which may have been due to this being a defective molecule or because of the model plants (N. benthamiana) used for the infection assays. Future experiments need to be conducted with other plants, including O. discolor, to understand more about the biology of these viral components.

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Quinolone-DNA Interaction: Sequence-Dependent Binding to Single-Stranded DNA Reflects the Interaction within the Gyrase-DNA Complex

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.3.854-862.2003 ◽

2003 ◽

Vol 47 (3) ◽

pp. 854-862 ◽

Cited By ~ 24

Author(s):

Christian G. Noble ◽

Faye M. Barnard ◽

Anthony Maxwell

Keyword(s):

Metal Ions ◽

Dna Cleavage ◽

Specific Binding ◽

Binding Mode ◽

Dna Interaction ◽

Dependent Manner ◽

Sequence Dependence ◽

Single Stranded Dna ◽

Dna Complex

ABSTRACT We have investigated the interaction of quinolones with DNA by a number of methods to establish whether a particular binding mode correlates with quinolone potency. The specificities of the quinolone-mediated DNA cleavage reaction of DNA gyrase were compared for a number of quinolones. Two patterns that depended on the potency of the quinolone were identified. Binding to plasmid DNA was examined by measuring the unwinding of pBR322 by quinolones; no correlation with quinolone potency was observed. Quinolone binding to short DNA oligonucleotides was measured by surface plasmon resonance. The quinolones bound to both single- and double-stranded oligonucleotides in an Mg2+-dependent manner. Quinolones bound to single-stranded DNA with a higher affinity, and the binding exhibited sequence dependence; binding to double-stranded DNA was sequence independent. The variations in binding in the presence of metal ions showed that Mg2+ promoted tighter, more specific binding to single-stranded DNA than softer metal ions (Mn2+ and Cd2+). Single-stranded DNA binding by quinolones correlated with the in vitro quinolone potency, indicating that this mode of interaction may reflect the interaction of the quinolone with DNA in the context of the gyrase-DNA complex.

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A Functional Histidine-Tagged Replication Initiator Protein: Implications for the Study of Single-Stranded DNA Virus Replication In Planta

Journal of Virology ◽

10.1128/jvi.79.13.8422-8430.2005 ◽

2005 ◽

Vol 79 (13) ◽

pp. 8422-8430 ◽

Cited By ~ 6

Author(s):

Julio C. Vega-Arreguín ◽

Tatiana Timchenko ◽

Bruno Gronenborn ◽

Bertha Cecilia Ramírez

Keyword(s):

Protein Complexes ◽

Affinity Purification ◽

Plant Viruses ◽

Replication Initiation ◽

In Planta ◽

Single Stranded Dna ◽

Rep Protein ◽

Initiator Protein ◽

Replication Initiator Protein ◽

Replication Initiator

ABSTRACT Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.

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Adeno-Associated Virus Rep Protein-Mediated Inhibition of Transcription of the Adenovirus Major Late Promoter In Vitro

Journal of Virology ◽

10.1128/jvi.00183-06 ◽

2006 ◽

Vol 80 (13) ◽

pp. 6207-6217 ◽

Cited By ~ 11

Author(s):

Patrick G. Needham ◽

John M. Casper ◽

Vivian Kalman-Maltese ◽

Kristin Verrill ◽

John David Dignam ◽

...

Keyword(s):

Gene Expression ◽

Transcription Initiation ◽

Nucleotide Binding ◽

Dnase I ◽

Tata Box ◽

Adeno Associated Virus ◽

Rep Protein ◽

Rep Proteins ◽

Gel Shift

ABSTRACT Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four AAV replication proteins (Rep78, Rep68, Rep52, and Rep40) are pleiotropic effectors of virus integration, replication, transcription, and virion assembly. These proteins exert effects on Ad gene expression and replication. In transient plasmid transfection assays, Rep proteins inhibit gene expression from a variety of transcription promoters. We have examined Rep protein-mediated inhibition of transcription of the Ad major late transcription promoter (AdMLP) in vitro. Rep78/68 are the strongest transcription suppressors and the purine nucleotide binding site in the Rep proteins, and by implication, the ATPase activity or conformational change induced by nucleotide binding is required for full repression. Rep52 has modest effects, and Rep40 exerts no significant effect on transcription. Rep78/68 and their N-terminal 225-residue domain bind to a 55-bp AdMLP DNA fragment in gel shift assays, suggesting that protein-DNA interactions are required for inhibition. This interaction was confirmed in DNase I protection assays and maps to a region extending from the TATA box to the transcription initiation site. Gel shift, DNase I, and chemical cross-linking assays with TATA box-binding protein (TBP) and Rep68 indicate that both proteins interact with each other and with the promoter at adjacent sites. The demonstration of Rep interaction with TBP and the AdMLP suggests that Rep78/68 alter the preinitiation complex of RNA polymerase II transcription. These observations provide new insight into the mechanism of Rep-mediated inhibition of gene expression.

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Escherichia coli Rep protein and helicase IV. Distributive single-stranded DNA-dependent ATPases that catalyze a limited unwinding reaction in vitro

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1992.tb17074.x ◽

1992 ◽

Vol 207 (2) ◽

pp. 479-485 ◽

Cited By ~ 11

Author(s):

Janet E. YANCEY-WRONA ◽

Edger R. WOOD ◽

James W. GEORGE ◽

Karen R. SMITH ◽

Steven W. MATSON

Keyword(s):

Escherichia Coli ◽

Single Stranded Dna ◽

Rep Protein ◽

Helicase Iv

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Identification of Cellular Proteins That Interact with the Adeno-Associated Virus Rep Protein

Journal of Virology ◽

10.1128/jvi.01939-08 ◽

2008 ◽

Vol 83 (1) ◽

pp. 454-469 ◽

Cited By ~ 39

Author(s):

Kevin Nash ◽

Weijun Chen ◽

Max Salganik ◽

Nicholas Muzyczka

Keyword(s):

Dna Replication ◽

Protein Complexes ◽

Protein Translation ◽

Cellular Protein ◽

Productive Infection ◽

Cellular Proteins ◽

Adeno Associated Virus ◽

Rep Protein ◽

Rep Proteins

ABSTRACT Adeno-associated virus (AAV) codes for four related nonstructural Rep proteins. AAV both replicates and assembles in the nucleus and requires coinfection with a helper virus, either adenovirus (Ad) or herpesvirus, for a productive infection. Like other more complex DNA viruses, it is believed that AAV interacts or modifies host cell proteins to carry out its infection cycle. To date, relatively little is known about the host proteins that interact with the viral Rep proteins, which are known to be directly involved in DNA replication, control of viral and cellular transcription, splicing, and protein translation. In this study, we used affinity-tagged Rep protein to purify cellular protein complexes that were associated with Rep in cells that had been infected with Ad and AAV. In all, we identified 188 cellular proteins from 16 functional categories, including 14 transcription factors, 6 translation factors, 15 potential splicing proteins, 5 proteins involved in protein degradation, and 13 proteins involved in DNA replication or repair. This dramatically increases the number of potential interactions over the current number of approximately 26. Twelve of the novel proteins found were further tested by coimmunoprecipitation or colocalization using confocal immunomicroscopy. Of these, 10 were confirmed as proteins that formed complexes with Rep, including proteins of the MCM complex (DNA replication), RCN1 (membrane transport), SMC2 (chromatin dynamics), EDD1 (ubiquitin ligase), IRS4 (signal transduction), and FUS (splicing). Computer analysis suggested that 45 and 28 of the 188 proteins could be placed in a pathway of interacting proteins involved in DNA replication and protein synthesis, respectively. Of the proteins involved in DNA replication, all of the previously identified proteins involved in AAV DNA replication were found, except Ad DBP. The only Ad protein found to interact with Rep was the E1b55K protein. In addition, we confirmed that Rep interacts with Ku70/80 helicase. In vitro DNA synthesis assays demonstrated that although Ku helicase activity could substitute for MCM to promote strand displacement synthesis, its presence was not essential. Our study suggests that the interaction of AAV with cellular proteins is much more complex than previously suspected and provides a resource for further studies of the AAV life cycle.

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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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The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III

Canadian Journal of Biochemistry ◽

10.1139/o79-105 ◽

1979 ◽

Vol 57 (6) ◽

pp. 855-866 ◽

Cited By ~ 13

Author(s):

Seishi Takahashi ◽

Christian Hours ◽

Alan Chu ◽

David T. Denhardt

Keyword(s):

Escherichia Coli ◽

Cell Extract ◽

Dna Helicase ◽

Protein Product ◽

Duplex Dna ◽

Replicative Form ◽

Single Stranded Dna ◽

Rep Protein ◽

E Coli

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes ([Formula: see text], fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, λp rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with λp rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of [Formula: see text] replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from [Formula: see text]-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.

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