Functional analysis of cis- and trans-acting replication factors of porcine circovirus type 1

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.     

Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli

A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct, a unit-length chimeric PCV1(Rep)/PCV2(Cap) genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1(Cap)/PCV2(Rep) genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE(1) Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep' protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep' protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.     

Determination of the origin cleavage and joining domain of geminivirus Rep proteins.

Replication of the single-stranded DNA genome of plant geminiviruses follows a rolling circle mechanism. It strictly depends on a 'rolling circle replication initiator protein', the M(r) 41 kDa viral Rep protein, encoded by the C1 or AC1 genes. Using wheat dwarf virus (WDV) and tomato yellow leaf curl virus (TYLCV) as examples, we show that not only the full-size Rep proteins, but also a putative 30 kDa translation product of WDV open reading frame C1-N as well as an artificially shortened 24 kDa Rep of TYLCV, cleave and join single-stranded origin DNA in vitro. Thus the pivotal origin recognition and processing activities of geminivirus Rep proteins must be mediated by the amino-terminal domain of Rep.     

Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2

Circoviruses are the smallest circular single-stranded DNA viruses able to replicate in mammalian cells. Essential to their replication is the replication initiator, or Rep protein that initiates the rolling circle replication (RCR) of the viral genome. Here we report the NMR solution three-dimensional structure of the endonuclease domain from the Rep protein of porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome in swine. The domain comprises residues 12-112 of the full-length protein and exhibits the fold described previously for the Rep protein of the representative geminivirus tomato yellow leaf curl Sardinia virus. The structure, however, differs significantly in some secondary structure elements that decorate the central five-stranded beta-sheet, including the replacement of a beta-hairpin by an alpha-helix in PCV2 Rep. The identification of the divalent metal binding site was accomplished by following the paramagnetic broadening of NMR amide signals upon Mn(2+) titration. The site comprises three conserved acidic residues on the exposed face of the central beta-sheet. For the 1:1 complex of the PCV2 Rep nuclease domain with a 22mer double-stranded DNA oligonucleotide chemical shift mapping allowed the identification of the DNA binding site on the protein and aided in constructing a model of the protein/DNA complex.     

Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus

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.     

Mapping and characterization of the origin of DNA replication of porcine circovirus.

The origin of DNA replication of porcine circovirus (PCV) was mapped to a 111-bp fragment. On top of a hairpin, a nonanucleotide (TAGTATTAC) homologous to nonanucleotides of other viruses was identified. Mutation of this element abolishes replication. PCV may be related to a virus family characterized by single-stranded circular DNA genomes, rolling-circle replication, and homology of their rep proteins.     

A biochemical characterization of the adeno-associated virus Rep40 helicase

The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replication (Rep), proteins. The Rep78 and Rep68 proteins regulate viral DNA replication, chromosomal integration, and gene expression. The Rep52 and Rep40 proteins mediate virus assembly. To better understand Rep protein function, we have expressed the Rep40 protein in Escherichia coli and purified it to near homogeneity. Like the other Rep proteins, Rep40 possesses helicase and ATPase activity. ATP is the best substrate, and Mg2+ is the most efficient divalent metal ion for helicase activity. A Lys to His mutation in the purine nucleotide-binding site results in a protein that inhibits helicase activity in a dominant negative manner. Rep40 unwinds double-stranded DNA containing a 3' single-stranded end, or blunt end, unlike the Rep68 and Rep52 enzymes, which have a strict requirement for DNA duplexes containing a 3' single-stranded end. Values for KATP in the ATPase assay are 1.1 +/- 0.2 mM and 1.2 +/- 0.2 mM in the absence and presence, respectively, of single-stranded DNA. Values for Vmax are 220 +/- 10 and 1,500 +/- 90 nmol/min/mg in the absence and presence, respectively, of single-stranded DNA. These studies provide the first enzymatic characterization of the AAV Rep40 protein and elucidate important functional differences between the AAV helicases.     

A single rep protein initiates replication of multiple genome components of faba bean necrotic yellows virus, a single-stranded DNA virus of plants

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 five rep 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.     

Solution structure of the endonuclease domain from the master replication initiator protein of the nanovirus faba bean necrotic yellows virus and comparison with the corresponding geminivirus and circovirus structures

Nanoviruses are a family of plant viruses that possess a genome of multiple circular single-stranded DNA (ssDNA) components and are strikingly similar in their replication mode to the plant geminiviruses and to the circoviruses that infect birds or mammals. These viruses multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyze the initiation of replication on a double-stranded DNA (dsDNA) intermediate and the resolution of the ssDNA into circles. Here we report the solution NMR three-dimensional structure of the endonuclease domain from the master Rep (M-Rep) protein of faba bean necrotic yellows virus (FBNYV), a representative of the nanoviruses. The domain comprises amino acids 2-95 (M-Rep2-95), and its global fold is similar to those previously described for the gemini- and circovirus Rep endonuclease domains, consisting of a central 5-stranded antiparallel beta-sheet covered on one side by an alpha-helix and irregular loops and on the other, more open side of the domain, by an alpha-helix containing the catalytic tyrosine residue (the catalytic helix). Longer domain constructs extending to amino acids 117 and 124 were also characterized. They contain an additional alpha-helix, are monomeric, and exhibit catalytic activity indistinguishable from that of M-Rep2-95. The binding site for the catalytic metal was identified by paramagnetic broadening and maps to residues on the exposed face of the central beta-sheet. A comparison with the previously determined Rep endonuclease domain structures of tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus, and that of porcine circovirus type 2 (PCV2) Rep allows the identification of a positively charged surface that is most likely involved in dsDNA binding, and reveals common features shared by all endonuclease domains of nanovirus, geminivirus, and circovirus Rep proteins.     

Creating a Novel Origin of Replication through Modulating DNA-Protein Interfaces

Background

While the molecular mechanisms of DNA-protein specificity at the origin of replication have been determined in many model organisms, these interactions remain unknown in the majority of higher eukaryotes and numerous vertebrate viruses. Similar to many viral origins of replication, adeno-associated virus (AAV) utilizes a cis-acting origin of replication and a virus specific Replication protein (Rep) to faithfully carry out self-priming replication. The mechanisms of AAV DNA replication are generally well understood. However, the molecular basis of specificity between the Rep protein and the viral origin of replication between different AAV serotypes remains uncharacterized.

Methodology/Principal Findings

By generating a panel of chimeric and mutant origins between two AAV serotypes, we have mapped two independent DNA-Protein interfaces involved in replicative specificity. In vivo replication assays and structural modeling demonstrated that three residues in the AAV2 Rep active site are necessary to cleave its cognate origin. An analogous origin (AAV5) possesses a unique interaction between an extended Rep binding element and a 49 aa region of Rep containing two DNA binding interfaces.

Conclusions/Significance

The elucidation of these structure-function relationships at the AAV origin led to the creation of a unique recombinant origin and compatible Rep protein with properties independent of either parent serotype. This novel origin may impact the safety and efficacy of AAV as a gene delivery tool. This work may also explain the unique ability of certain AAV serotypes to achieve site-directed integration into the human chromosome. Finally, this result impacts the study of conserved DNA viruses which employ rolling circle mechanisms of replication.     

Herpes simplex virus type 1 ICP8: helix-destabilizing properties.

The major single-stranded DNA-binding protein, ICP8, of herpes simplex virus type 1 (HSV-1) is one of seven virus-encoded polypeptides required for HSV-1 DNA replication. To investigate the role of ICP8 in viral DNA replication, we have examined the interaction of ICP8 with partial DNA duplexes and found that it can displace oligonucleotides annealed to single-stranded M13 DNA. In addition, ICP8 can melt small fragments of fully duplex DNA. Unlike a DNA helicase, ICP8-promoted strand displacement is ATP and Mg2+ independent and exhibits no directionality. It requires saturating amounts of ICP8 and is both efficient and highly cooperative. These properties make ICP8 suitable for a role in DNA replication in which ICP8 destabilizes duplex DNA during origin unwinding and replication fork movement.     

An adeno-associated virus (AAV) initiator protein, Rep78, catalyzes the cleavage and ligation of single-stranded AAV ori DNA

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., Mn(2+) or Mg(2+)) 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.     

Cellular Proteins Required for Adeno-Associated Virus DNA Replication in the Absence of Adenovirus Coinfection

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3′ primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase α-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase δ and , we attempted to reconstitute AAV DNA replication by substituting either purified polymerase δ or polymerase for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.     

Interaction between a geminivirus replication protein and the plant sumoylation system

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells after accumulation of host replication machinery. Tomato golden mosaic virus (TGMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) encode a protein, RepAC1 (or Rep), that is essential for viral replication. Rep/RepAC1 is an oligomeric protein that binds to double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and is sufficient for host induction. It also interacts with several host proteins, including the cell cycle regulator, retinoblastoma, and essential components of the cell DNA replication machinery, like proliferating nuclear cell antigen (PCNA) and RFC-1. To identify other cellular proteins that interact with Rep/RepAC1 protein, a Nicotiana benthamiana cDNA library was screened with a yeast two-hybrid assay. The host cell sumoylation enzyme, NbSCE1 (N. benthamiana SUMO-conjugating enzyme, homolog to Saccharomyces cerevisiae UBC9), was found to interact specifically with RepAC1. Mapping studies localized the interaction to the N-terminal half of RepAC1. Effects on geminivirus replication were observed in transgenic plants with altered levels of SUMO, the substrate for UBC9.     

Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile.

The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the double-stranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.     

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase δ was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.Adeno-associated virus (AAV) is a human parvovirus that is currently used as a gene transfer vector (14). AAV particles consist of a small icosahedral capsid protecting a single 4.7-kb single-stranded DNA (ssDNA) genome with two open reading frames, rep and cap, surrounded by inverted terminal repeats (ITRs). The ITRs are the only sequences required in cis for genome replication and packaging. The rep gene encodes four nonstructural Rep proteins: Rep78, -68, -52, and -40. The two larger isoforms, Rep78 and -68, have origin binding, helicase, and site-specific endonuclease activities and are involved in AAV gene expression and genome processing, including replication and site-specific integration (39). The two smaller Rep isoforms are not required for AAV DNA replication but are involved in the control of viral gene expression and packaging of viral DNA (30).When wild-type (wt) AAV infects a cell in the absence of a helper virus, it enters latency. Latent AAV genomes persist in cells either as episomes or as integrated genomes, preferentially at a specific locus (named AAVS1) on human chromosome 19. In most instances, no detectable viral gene expression or genome replication occurs unless the cell is co- or superinfected by a helper virus, such as adenovirus, herpes simplex virus type 1 (HSV-1), or HSV-2. Under these conditions, AAV replication and assembly take place in large intranuclear domains called replication compartments (RCs) that frequently colocalize with replication domains formed by the helper virus itself (81). The viral genome replicates by leading-strand synthesis and generates new ssDNA molecules by a strand displacement mechanism that occurs after strand- and site-specific cleavage of viral DNA by Rep78/68 within the ITRs (39).Studies conducted on the relationship between AAV and its helper viruses are important not only to identify helper activities that can be used to produce recombinant AAV vectors but also to understand how AAV adapts its replication strategy to the helper virus and to the nuclear environment in general. Adenovirus helper functions have historically been the first and most extensively studied functions. These studies have shown that adenovirus helps AAV by stimulating viral gene expression and by enhancing AAV genome replication, mostly indirectly (19). Indeed, early studies showed that the adenovirus polymerase (E2b) is dispensable for AAV replication (8) and that the viral DNA-binding protein (DBP), the product of the E2a gene, is able to modestly enhance the processivity of AAV genome replication in vitro (77). More recently, the adenovirus proteins E1b55k and E4orf6 were shown to stimulate AAV genome replication by degrading the cellular Mre11/Rad50/Nbs1 (MRN) complex that restricts AAV genome replication during adenovirus coinfection (32). The concept that AAV genome replication can rely mostly, if not uniquely, on direct help from cellular factors was further strengthened by the demonstration that purified proteins such as replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), minichromosome maintenance (MCM) proteins, and DNA polymerase δ (Pol δ) were sufficient to replicate the AAV genome in vitro in the presence of Rep (40-41, 43). The involvement of these cellular proteins during AAV genome replication was also confirmed by the proteomic analysis of factors associated with Rep proteins during adenovirus-induced AAV replication (42).Interestingly, studies conducted on HSV-1 helper activities suggest that the strategy of AAV replication may vary depending on the helper virus. Indeed, previous studies showed that the HSV-1 helicase-primase (HP) complex (UL5/8/52) and DBP (ICP8) could replicate transfected AAV-2 plasmids (80) and that the helicase activity, but not primase activity, of the HP complex was required for this effect (62, 66). More recently, a comprehensive study of HSV-1 helper activities demonstrated that the HSV-1 immediate-early proteins ICP0, ICP4, and ICP22 could stimulate rep gene expression, probably by diminishing intrinsic antiviral effects (1, 18). In addition, the HSV-1 DNA polymerase encoded by UL30, along with its associated processivity factor (UL42), although not strictly required, was demonstrated to significantly increase AAV replication levels induced in the presence of the HP complex and ICP8. Interestingly, the HSV-1 HP complex, DBP, and polymerase were also shown to be sufficient to replicate AAV DNA in vitro in the presence of Rep proteins without any cellular protein (78). Altogether, these observations indicate that in the context of an HSV-1 coinfection, AAV relies extensively on viral activities provided by the helper that directly participate in AAV genome replication.To further elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis to identify the cellular and HSV-1 factors associated with Rep proteins and, consequently, potentially recruited within AAV RCs. To analyze Rep-associated proteins in the presence and absence of HSV-1 DNA replication, this analysis was performed using wt HSV-1 and an HSV-1 mutant in which the DNA polymerase encoded by the UL30 gene is absent (HSVΔUL30). This study resulted in the identification of approximately 60 cellular proteins, among which the largest functional categories corresponded to factors involved in DNA and RNA metabolism. Immunofluorescence analyses confirmed that in the presence of HSV-1, a basal set of cellular DNA replication enzymes, including RPA, RFC, and PCNA, was recruited within AAV RCs, with the exception of the MCM helicases. The cellular DNA polymerases, in particular Pol δ, were not identified by this analysis but subsequently were shown to be recruited in AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, our results indicate that AAV replication induced by HSV-1 is associated with the recruitment of DNA repair factors, including components of the MRN complex, the Ku proteins, PARP-1, and factors of the mismatch repair (MMR) pathway. Finally, several HSV-1 proteins, most notably the UL12 protein, were also identified within AAV RCs. Our analyses confirmed the association between UL12 and Rep and demonstrated for the first time that this viral exonuclease plays a critical role during AAV replication by enhancing the formation of discrete AAV replicative forms and the production of AAV particles.Altogether, these results indicate that in the presence of HSV-1, AAV may replicate by using a basal set of cellular DNA replication enzymes but also relies extensively on HSV-1-derived proteins for its replication, including UL12, a newly discovered helper factor. These results suggest that AAV may be able to differentially adapt its replication strategy to the nuclear environment induced by the helper virus.     

Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model

Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.