scholarly journals Demonstration of Nicking/Joining Activity at the Origin of DNA Replication Associated with the Rep and Rep′ Proteins of Porcine Circovirus Type 1

2006 ◽  
Vol 80 (13) ◽  
pp. 6225-6234 ◽  
Author(s):  
Tobias Steinfeldt ◽  
Tim Finsterbusch ◽  
Annette Mankertz
Keyword(s):  
Dna Replication ◽  
Virus Replication ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Loop Structure ◽  
Stem Loop ◽  
Single Strand Break ◽  
Stem Loop Structure ◽  
Rep Proteins

ABSTRACT The replication of porcine circovirus type 1 (PCV1) is thought to occur by rolling-circle replication (RCR), whereby the introduction of a single-strand break generates a free 3′-hydroxyl group serving as a primer for subsequent DNA synthesis. The covalently closed, single-stranded genome of PCV1 replicates via a double-stranded replicative intermediate, and the two virus-encoded replication-associated proteins Rep and Rep′ have been demonstrated to be necessary for virus replication. However, although postulated to be involved in RCR-based virus replication, the mechanism of action of Rep and Rep′ is as yet unknown. In this study, the ability of PCV1 Rep and Rep′ to “nick” and “join” strand discontinuities within synthetic oligonucleotides corresponding to the origin of replication of PCV1 was investigated in vitro. Both proteins were demonstrated to be able to cleave the viral strand between nucleotides 7 and 8 within the conserved nonanucleotide motif (5′-TAGTATTAC-3′) located at the apex of a putative stem-loop structure. In addition, the Rep and Rep′ proteins of PCV1 were demonstrated to be capable of joining viral single-stranded DNA fragments, suggesting that these proteins also play roles in the termination of virus DNA replication. This joining activity was demonstrated to be strictly dependent on preceding substrate cleavage and the close proximity of origin fragments accomplished by base pairing in the stem-loop structure. The dual “nicking/joining” activities associated with PCV1 Rep and Rep′ are pivotal events underlying the RCR-based replication of porcine circoviruses in mammalian cells.

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

2007 ◽  
Vol 81 (11) ◽  
pp. 5696-5704 ◽  
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.

scholarly journals Mechanism of Rep-Mediated Adeno-Associated Virus Origin Nicking

2000 ◽  
Vol 74 (17) ◽  
pp. 7762-7771 ◽  
Author(s):  
J. Rodney Brister ◽  
Nicholas Muzyczka
Keyword(s):  
Specific Activity ◽  
Dna Helicase ◽  
Loop Structure ◽  
Stem Loop ◽  
Adeno Associated Virus ◽  
Stem Loop Structure ◽  
Virus Origin ◽  
Rep Proteins ◽  
Sequence Elements ◽  
Origin Function

ABSTRACT The single-stranded adeno-associated virus type 2 (AAV) genome is flanked by terminal repeats (TRs) that fold back on themselves to form hairpinned structures. During AAV DNA replication, the TRs are nicked by the virus-encoded Rep proteins at the terminal resolution site (trs). This origin function apparently requires three sequence elements, the Rep binding element (RBE), a small palindrome that comprises a single tip of an internal hairpin within the TR (RBE′), and the trs. Previously, we determined the sequences at the trs required for Rep-mediated cleavage and demonstrated that the trs endonuclease reaction occurs in two discrete steps. In the first step, the Rep DNA helicase activity unwinds the TR, thereby extruding a stem-loop structure at thetrs. In the second step, Rep transesterification activity cleaves the trs. Here we investigate the contribution of the RBE and RBE′ during this process. Our data indicate that Rep is tethered to the RBE in a specific orientation duringtrs nicking. This orientation appears to align Rep on the AAV TR, allowing specific nucleotide contacts with the RBE′ and directing nicking to the trs. Accordingly, alterations in the polarity or position of the RBE relative to the trsgreatly inhibit Rep nicking. Substitutions within the RBE′ also reduce Rep specific activity, but to a lesser extent. Interestingly, Rep interactions with the RBE and RBE′ during nicking seem to be functionally distinct. Rep contacts with the RBE appear necessary for both the DNA helicase and trs cleavage steps of the endonuclease reaction. On the other hand, RBE′ contacts seem to be required primarily for TR unwinding and formation of thetrs stem-loop structure, not cleavage. Together, these results suggest a model of Rep interaction with the AAV TR during origin nicking through a tripartite cleavage signal comprised of the RBE, the RBE′, and the trs.

scholarly journals Human Cytomegalovirus UL84 Interacts with an RNA Stem-Loop Sequence Found within the RNA/DNA Hybrid Region of oriLyt

2007 ◽  
Vol 81 (13) ◽  
pp. 7077-7085 ◽  
Author(s):  
Kelly S. Colletti ◽  
Kate E. Smallenburg ◽  
Yiyang Xu ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Specific Interaction ◽  
Simian Virus 40 ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Core Proteins ◽  
Essential Region

ABSTRACT Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the complex cis-acting oriLyt region, which spans nearly 3 kb. DNA synthesis requires six core proteins together with UL84 and IE2. Previously, two essential regions were identified within oriLyt. Essential region I (nucleotides [nt] 92209 to 92573) can be replaced with the constitutively active simian virus 40 promoter, which in turn eliminates the requirement for IE2 in the origin-dependent transient-replication assay. Essential region II (nt 92979 to 93513) contains two elements of interest: an RNA/DNA hybrid domain and an inverted repeat sequence capable of forming a stem-loop structure. Our studies now reveal for the first time that UL84 interacts with a stem-loop RNA oligonucleotide in vitro, and although UL84 interacted with other nucleic acid substrates, a specific interaction occurred only with the RNA stem-loop. Increasing concentrations of purified UL84 produced a remarkable downward-staircase pattern, which is not due to a nuclease activity but is dependent upon the presence of secondary structures, suggesting that UL84 modifies the conformation of the RNA substrate. Cross-linking experiments show that UL84 possibly changes the conformation of the RNA substrate. The addition of purified IE2 to the in vitro binding reaction did not affect binding to the stem-loop structure. Chromatin immunoprecipitation assays performed using infected cells and purified virus show that UL84 is bound to oriLyt in a region adjacent to the RNA/DNA hybrid and the stem-loop structure. These results solidify UL84 as the potential initiator of HCMV DNA replication through a unique interaction with a conserved RNA stem-loop structure within oriLyt.

scholarly journals Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome

2017 ◽  
Vol 65 (2) ◽  
pp. 278-290 ◽  
Author(s):  
Jie Cai ◽  
Xiaohong Xie ◽  
Yi Hu ◽  
Yang Zhan ◽  
Wanting Yu ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Recombinant Baculovirus ◽  
Porcine Circovirus ◽  
Economic Losses ◽  
Loop Structure ◽  
Swine Industry ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Pcv2 Replication ◽  
Pcv2 Genome

Porcine circovirus associated diseases (PCVAD) caused by PCV2 are responsible for severe economic losses in the swine industry. The mechanism of PCV2 replication has not been fully elucidated yet. PCV2 may be successfully rescued by means of either an infectious DNA clone containing the full length of the viral genomic DNA, or from PCV2-infected clinical tissues in PK15 cell culture. However, viruses harvested by both methods have low titres. In this study, PCV2 was prepared with a higher titre from PK15 cells infected by recombinant baculoviruses containing 1PCV2 (one stem-loop structure) or 1.1PCV2 (two stem-loop structure) genomic DNA copy. In addition, infectious DNA clones containing two stem-loop structures in either plasmid or baculovirus backbones are capable of generating a higher virus titre than the DNA clones with only one copy of stem-loop structure.

scholarly journals The Human Immunodeficiency Virus Type 1 TAR RNA Upper Stem-Loop Plays Distinct Roles in Reverse Transcription and RNA Packaging

2000 ◽  
Vol 74 (12) ◽  
pp. 5639-5646 ◽  
Author(s):  
David Harrich ◽  
C. William Hooker ◽  
Emma Parry
Keyword(s):  
Reverse Transcription ◽  
Transcription Initiation ◽  
Loop Structure ◽  
Rna Packaging ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Immunodeficiency Virus ◽  
Tar Rna ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) RNA genome is flanked by a repeated sequence (R) that is required for HIV-1 replication. The first 57 nucleotides of R form a stable stem-loop structure called the transactivation response element (TAR) that can interact with the virally encoded transcription activator protein, Tat, to promote high levels of gene expression. Recently, we demonstrated that TAR is also important for efficient HIV-1 reverse transcription, since HIV-1 mutated in the upper stem-loop of TAR showed a reduced ability both to initiate and to complete reverse transcription. We have analyzed a series of HIV-1 mutant viruses to better defined the structural or sequence elements required for natural endogenous reverse transcription and packaging of virion RNA. Our results indicate that the requirement for TAR in reverse transcription is conformation dependent, since mutants with mutations that alter the upper stem-loop orientation are defective for reverse transcription initiation and have minor defects in RNA packaging. In contrast, TAR mutations that allowed the formation of alternative upper stem-loop structure greatly reduced RNA packaging but did not affect reverse transcription efficiency. These results are consistent with direct involvement of the upper stem-loop structure in packaging of genomic RNA and suggest that the TAR RNA stem-loop from nucleotide +18 to +42 interacts with other components of the reverse transcription initiation complex to promote efficient reverse transcription.

scholarly journals Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

2017 ◽  
Vol 13 (4) ◽  
pp. e1006296 ◽  
Author(s):  
Yuanjie Liu ◽  
Hui Nie ◽  
Richeng Mao ◽  
Bidisha Mitra ◽  
Dawei Cai ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Viral Rna ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
B Virus

scholarly journals Crystal Structure of the Dimerized N Terminus of Porcine Circovirus Type 2 Replicase Protein Reveals a Novel Antiviral Interface

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Gao Luo ◽  
Xiongfei Zhu ◽  
Yang Lv ◽  
Bomin Lv ◽  
Jin'e Fang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Virus Replication ◽  
Hydrophobic Interactions ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Protein Dimerization ◽  
Rep Proteins ◽  
N Terminus

ABSTRACT Two replicase (Rep) proteins, Rep and Rep′, are encoded by porcine circovirus (PCV) ORF1; Rep is a full ORF1 transcript, and Rep′ is a truncated transcript generated by splicing. These two proteins are crucial for the rolling-circle replication (RCR) of PCV. The N-terminal sequences of Rep and Rep′ are identical and interact to form homo- or heterodimers. The three types of dimers perform different functions during replication. A structural examination of the interfacing termini has not been performed. In this study, a crystal structure of dimerized Rep protein N termini was resolved at 2.7 Å. The dimerized protein was maintained by nine intermolecular hydrogen bonds and 15 pairs of hydrophobic interactions. The amino acid residue Ile37 participates in 11 of the hydrophobic interactions, mostly with its side chain. To find the predominant sites for protein dimerization and virus replication, a series of mutant proteins and virus replicons were generated by alanine substitution. Of all the single amino acid substitutions, the mutation at Ile37 showed the greatest effect on protein dimerization and virus replication. A double mutation at Leu35 and Ile37 almost eliminated protein dimerization and had the greatest negative effect on virus replication. These studies demonstrate that Leu35 and Ile37 are the most important residues for protein dimerization and are crucial for virus replication. Our results also show that PCV replication can be decreased by disrupting the dimerization of Rep or Rep′ at the N terminus, suggesting that the structural interface responsible for dimerization offers a promising antiviral target. IMPORTANCE Porcine circovirus type 2 (PCV2) is one of the most economically damaging pathogens affecting the swine industry. Although vaccines have been available for more than 10 years, the virus still remains prevalent. More effective strategies for disease prevention are clearly required. The Rep and Rep′ proteins of the virus have identical N-terminal regions that interact with each other, allowing the formation of homo- or heterodimers. The heterodimer has crucial functions during different stages of viral replication. Here, we resolved the crystal structure of the Rep (Rep′) dimerization domain. The individual residues involved in the intermolecular interaction were visualized in the protein structure, and several interactions were verified by mutant analysis. Our studies show that disrupting the interaction decreases viral replication, thus revealing a new target for the design of antiviral agents.

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