Search

Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic.

The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication.

Giant Viruses are a widespread group of viruses, characterized by huge genomes composed of a small subset of ancestral, vertically inherited core genes along with a large body of highly variable genes. In this study, I report the acquisition of 23 core ancestral Giant Virus genes by diverse eukaryotic species including various protists, a moss and a cnidarian. The viral genes are inserted in large scaffolds or chromosomes with intron-rich, eukaryotic-like genomic contexts, refuting the possibility of DNA contaminations. Some of these genes are expressed and in the cryptophyte alga Guillardia theta, a possible non-homologous displacement of the eukaryotic DNA primase by a viral D5 helicase/primase is documented. As core Giant Virus genes represent only a tiny fraction of the total genomic repertoire of these viruses, these results suggest that Giant Viruses represent an underestimated source of new genes and functions for their hosts.

JC virus (JCV) lytically infects the oligodendrocytes in the central nervous system in a subset of immunocompromized patients and causes the demyelinating disease, progressive multifocal leukoencephalopathy. JCV replicates and assembles into infectious virions in the nucleus. However, understanding the molecular mechanisms of its virion biogenesis remains elusive. In this report, we have attempted to shed more light on this process by investigating molecular interactions between large T antigen (LT-Ag), Hsp70 and minor capsid proteins, VP2/VP3. We demonstrated that Hsp70 interacts with VP2/VP3 and LT-Ag; and accumulates heavily in the nucleus of the infected cells. We also showed that VP2/VP3 associates with LT-Ag through their DNA binding domains resulting in enhancement in LT-Ag DNA binding to Ori and induction in viral DNA replication. Altogether, our results suggest that VP2/VP3 and Hsp70 actively participate in JCV DNA replication and may play critical roles in coupling of viral DNA replication to virion encapsidation.

The non-structural protein 1 (NS1) of human parvovirus B19 plays a critical role in viral DNA replication. Previous studies identified the origin of replication in the viral DNA, which contains four DNA elements, namely NSBE1 to NSBE4, that are required for optimal viral replication (Guan et al, 2009, J. Virology, 83, 9541-9553). Here we have demonstrated in vitro that the NS1 N-terminal domain (NS1N) binds to the origin of replication in a sequence-specific, length-dependent manner that requires NSBE1 and NSBE2, while NSBE3 and NSBE4 are dispensable. Mutagenesis analysis has identified nucleotides in NSBE1 and NSBE2 that are critical for NS1N binding. These results suggest that NS1 binds to the NSBE1-NSBE2 region in the origin of replication, while NSBE3 and NSBE4 may provide binding sites for potential cellular factors. Such a specialized nucleoprotein complex may enable NS1 to nick the terminal resolution site and separate DNA strands during replication.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication.

Productive replication of human papillomaviruses (HPV) is restricted to the uppermost layers of the differentiating epithelia. How HPV ensures an adequate supply of cellular substrates for viral DNA synthesis in a differentiating environment is unclear. Here, we demonstrate that HPV31 positive cells exhibit increased dNTP pools and levels of RRM2, a component of the ribonucleotide reductase (RNR) complex, which is required for de novo synthesis of dNTPs. RRM2 depletion blocks productive replication, suggesting RRM2 provides dNTPs for viral DNA synthesis in differentiating cells. We demonstrate that HPV31 regulates RRM2 levels through expression of E7 and activation of the ATR-Chk1-E2F1 DNA damage response, which is essential to combat replication stress upon entry into S-phase, as well as for productive replication. Our findings suggest a novel way in which viral DNA synthesis is regulated through activation of ATR and Chk1 and highlight an intriguing new virus/host interaction utilized for viral replication.

Integration of human papillomaviruses into that of the host promotes genomic instability and progression to cancer; factors that promote integration remain to be fully identified. DNA damage agents can promote double strand breaks during DNA replication providing substrates for integration and we investigated the ability of DNA damage to regulate HPV E1 and E2 mediated DNA replication. Results demonstrate that HPV E1 and E2 replication is not arrested following DNA damage, both in vivo and in vitro, while replication by SV40 Large T antigen is arrested and ATR is the candidate kinase for mediating the arrest. LTAg is a target for PIKK DNA damage signalling kinases, while E1 is not. We propose that the failure of E1 to be targeted by PIKKs allows HPV replication in the presence of DNA damaging agents. Such replication will result in double strand breaks in the viral genome ultimately promoting viral integration and cervical cancer.

The human papillomavirus 16 E2 protein regulates transcription from, and replication of, the viral genome and is also required for segregation of the viral genome via interaction with mitotic bodies. To regulate DNA replication E2 interacts with sequences around the origin of replication and recruits the viral helicase E1 via a protein-protein interaction, which then initiates viral genome replication. The replication role of E2 must originally function in a host cell S phase. In this report, we demonstrate that E2 is stabilised in the S phase of the cell cycle and that this stabilisation is accompanied by an increase in phosphorylation of the protein. This increased phosphorylation and stability are likely required for optimum viral DNA replication and therefore identification of the enzymes involved in regulating these properties of E2 will provide targets for therapeutic intervention in the viral life cycle. Preliminary studies have identified E2 as a Cdk2 substrate demonstrating this enzyme as a candidate kinase for mediating the in vivo phosphorylation of HPV16 E2.

In order to explore the mechanism of geminivirus DNA replication, we show that the Replication initiator (Rep) protein encoded by Mungbean yellow mosaic India virus (MYMIV), a member of the family Geminiviridae, binds specifically to the iterons present in the viral DNA replication origin (CR-A) in a highly ordered manner that might be a prerequisite for the initiation of replication. MYMIV Rep also acts as a helicase during the post-initiation stage and is upregulated in presence of the RPA32 subunit of Replication Protein A. The implication of these findings on the initiation and elongation stages of MYMIV DNA replication has been discussed.

Adenovirus (Ad) infections stimulate the activation of cellular DNA damage response and repair pathways. Ad early regulatory proteins prevent activation of DNA damage responses by targeting the MRN complex, composed of the Mre11, Rad50 and Nbs1 proteins, for relocalization and degradation. In the absence of these viral proteins, Mre11 colocalizes with viral DNA replication foci. Mre11 foci formation at DNA damage induced by ionizing radiation depends on the Nbs1 component of the MRN complex and is stabilized by the mediator of DNA damage checkpoint protein 1 (Mdc1). We find that Nbs1 is required for Mre11 localization at DNA replication foci in Ad E4 mutant infections. Mre11 is important for Mdc1 foci formation in infected cells, consistent with its role as a sensor of DNA damage. Chromatin immunoprecipitation assays indicate that both Mre11 and Mdc1 are physically bound to viral DNA, which could account for their localization in viral DNA containing foci. Efficient binding of Mre11 to E4 mutant DNA depends on the presence of Nbs1, and is correlated with a significant E4 mutant DNA replication defect. Our results are consistent with a model in which physical interaction of Mre11 with viral DNA is mediated by Nbs1, and interferes with viral DNA replication.

A stem–loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. In this work, a head-to-tail tandem construct of porcine circovirus capable of generating unit-length genomic DNA in Escherichia coli was employed to examine the role of the stem–loop structure with respect to the RCR initiation and termination process. The advantage of using a head-to-tail tandem construct is that the initiation and termination sites for generation of the unit-length viral genomes are physically separated, which allows independent examination of the initiation/termination processes. Nucleotide substitution mutational analysis showed that a pair of inverted repeats capable of forming a stem–loop structure was essential for termination, but not for initiation. The results also demonstrated that it is the stem–loop configuration, not nucleotide sequence specificity, that is critical for terminating RCR DNA replication.

In addition to their encapsidation function, viral coat proteins (CP) contribute to viral life cycle in many different ways. The CPs of the geminiviruses are responsible for intra- as well as inter-plant virus transmission and might determine the yield of viral DNA inside the infected tissues by either packaging the viral DNA or interfering with the viral replicative machinery. Since the cognate Rep largely controls the rolling circle replication of geminiviral DNA, the interaction between Rep and CP might be worthwhile to examine for elucidation of CP-mediated control of the viral DNA copy number. Here a reasonably strong interaction between Rep and CP of the geminivirus Mung bean yellow mosaic India virus is reported. The domain of interaction has been mapped to a central region of Rep. The replication initiation activity of Rep, i.e., its nicking and closing function, is down regulated by CP. This report highlights how CP could be important in controlling geminiviral DNA replication.

IE-1, LEF-3, and P143 are three of six proteins encoded by Autographa californica nucleopolyhedrovirus (AcMNPV) essential for baculovirus DNA replication in transient replication assays. IE-1 is the major baculovirus immediate early transcription regulator. LEF-3 is a single-stranded DNA binding protein (SSB) and P143 is a DNA helicase protein. To investigate their interactions in vivo, we treated AcMNPV-infected Spodoptera frugiperda cells with formaldehyde and separated soluble proteins from chromatin by cell fractionation and cesium chloride equilibrium centrifugation. Up to 70% of the total LEF-3 appeared in the fraction of soluble, probably nucleoplasmic proteins, while almost all P143 and IE-1 were associated with viral chromatin in the nucleus. This suggests that LEF-3 is produced in quantities that are higher than needed for the coverage of single stranded regions that arise during viral DNA replication and is consistent with the hypothesis that LEF-3 has other functions such as the localization of P143 to the nucleus. Using a chromatin immunoprecipitation procedure, we present the first direct evidence of LEF-3, P143, and IE-1 proteins binding to closely linked sites on viral chromatin in vivo, suggesting that they may form replication complexes on viral DNA in infected cells.

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein–Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection–replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP).

Herpes simplex virus (HSV) uses intranuclear compartmentalization to concentrate the viral and cellular factors required for the progression of the viral life cycle. Processes as varied as viral DNA replication, late gene expression, and capsid assembly take place within discrete structures within the nucleus called replication compartments. Replication compartments are hypothesized to mature from a few distinct structures, called prereplicative sites, that form adjacent to cellular nuclear matrix-associated ND10 sites. During productive infection, the HSV single-stranded DNA-binding protein ICP8 localizes to replication compartments. To further the understanding of replication compartment maturation, we have constructed and characterized a recombinant HSV-1 strain that expresses an ICP8 molecule with green fluorescent protein (GFP) fused to its C terminus. In transfected Vero cells that were infected with HSV, the ICP8–GFP protein localized to prereplicative sites in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA) or to replication compartments in the absence of PAA. A recombinant HSV-1 strain expressing the ICP8–GFP virus replicated in Vero cells, but the yield was increased by 150-fold in an ICP8-complementing cell line. Using the ICP8–GFP protein as a marker for replication compartments, we show here that these structures start as punctate structures early in infection and grow into large, globular structures that eventually fill the nucleus. Large replication compartments were formed by small structures that either moved through the nucleus to merge with adjacent compartments or remained relatively stationary within the nucleus and grew by accretion and fused with neighboring structures.

The UL84 gene of human cytomegalovirus (HCMV) is thought to be involved in the initiation of viral DNA replication, and is essential for replication of strains AD169 and Towne. Hence, discovery that strain TB40-BAC4 is viable in the absence of UL84 presented an enigma requiring an explanation. Data reported here show that strain TR also tolerated loss of UL84, whereas strains FIX, Merlin, Ph, and Toledo did not. UL84-independent growth required the viral replication origin. The genetic locus in TB40 that controls UL84 dependence was mapped to codon 388 of the UL122 gene, which encodes the immediate early 2 (IE2) 86kD protein. Introduction of this TB40-BAC4 variant (H388D) into FIX and Toledo clones converted these strains to UL84 independence. These results provide genetic evidence in virus-infected cells that supports the hypothesis that UL122 participates in the initiation of viral DNA replication by a mechanism involving transcription-mediated activation of the origin.

Geminiviruses replicate their small, single-stranded DNA genomes through double-stranded DNA intermediates in plant nuclei using host replication machinery. Like most dicot-infecting geminiviruses, tomato golden mosaic virus encodes a protein, AL3 or C3, that greatly enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that AL3 forms oligomers and interacts with the viral replication initiator AL1. Experiments reported here established that AL3 also interacts with a plant homolog of the mammalian tumor suppressor protein, retinoblastoma (pRb). Analysis of truncated AL3 proteins indicated that pRb and AL1 bind to similar regions of AL3, whereas AL3 oligomerization is dependent on a different region of the protein. Analysis of truncated AL1 proteins located the AL3-binding domain between AL1 amino acids 101 and 180 to a region that also includes the AL1 oligomerization domain and the catalytic site for initiation of viral DNA replication. Interestingly, the AL3-binding domain was fully contiguous with the domain that mediates AL1/pRb interactions. The potential significance of AL3/pRb binding and the coincidence of the domains responsible for AL3, AL1, and pRb interactions are discussed.

The Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA-1) is required for the maintenance of the viral chromosome in latently infected, proliferating cells and plays a role in latent cycle DNA replication. EBNA-1 also functions as a positive and negative regulator of EBV gene expression. We have investigated the interaction of EBNA-1 with p32, a host mitochondrial protein that associates with EBNA-1 in EBV-positive Burkitt's lymphoma cells. Using a chromatin immunoprecipitation assay, we found that a fraction of p32 localizes to the viral latent cycle origin of DNA replication oriP in vivo. p32 binds EBNA-1 independently of other proteins or DNA. EBNA-1 variants lacking one of two p32 binding elements did not interact stably with p32 in cultured cells and were defective for both transcriptional activation of a reporter gene linked to oriP FR and replication and/or maintenance of a plasmid bearing oriP. These results support a role for p32 in transcriptional activation by EBNA-1 and suggest that p32 plays a role in EBV latent cycle DNA replication.

We have characterized two polyomavirus large T antigen mutants with different properties in viral DNA replication. dl-97, a mutant active in immortalization, exerts a dominant negative effect in viral DNA replication. 13val, which is defective in both immortalization and viral DNA replication, has a lesion in the putative DnaJ domain affecting the block of Rb function.

Eukaryotic DNA is organized into domains or loops generated by the attachment of chromatin fibers to the nuclear matrix via specific regions called scaffold or matrix attachment regions. The role of these regions in DNA replication is currently under investigation since they have been found in close association with origins of replication. Also, viral DNA sequences, containing the origins of replication, have been found attached to the nuclear matrix. To investigate the functional role of this binding we have studied, in Raji cells, the interaction between Epstein–Barr virus (EBV) origins of replication and the nuclear matrix in relation to the viral cycle of infection. We report here that both the latent (ori P) and the lytic (ori Lyt) EBV origins of replication are attached to the nuclear matrix, the first during the latent cycle of infection and the second after induction of the lytic cycle. These findings suggest that the binding of the origins of replication with the nuclear matrix modulates viral replication and expression in the two different phases of infection.

Autographa californicamulticapsid nucleopolyhedrovirus (AcMNPV) encodes a 143-kDa protein (P143) required for viral DNA synthesis and involved in host range determination. The predicted amino acid sequence of P143 contains seven motifs (I, Ia, II–VI) shared with a superfamily of helicases involved in the unwinding of duplex nucleic acids; a putative DNA binding motif; a putative nuclear localization signal (NLS); and a demonstrated host range motif. In this study, the functional significance of these conserved P143 motifs was examined by site-specific mutation resulting in amino acid substitutions of conserved residues within each of them. Anin vivocomplementation replication assay was developed and each mutated P143 protein expressed from a transfected plasmid was tested for its ability to complement the replication-negative ts8 baculovirus mutant for the amplification of an origin-containing plasmid. Mutations in the helicase motifs I, Ia, and II and in a potential helix-turn-helix motif abolished the ability of P143 to complement the ts8 defect in DNA replication, suggesting that these conserved amino acid residues may be essential for the replication function of the protein. In contrast, mutation of conserved amino acid residues in the helicase motifs IV, V, and VI did not affect the ability of the P143 proteins to complement the replication defect of ts8. A mutation in motif III caused a reduction in the replication function of P143. Deletion of Gly552 in the host range region eliminated the replication function of P143. Mutations within a putative NLS had no effect on the ability of P143 to support DNA replication, suggesting that these residues are nonessential and that the putative P143 NLS sequence may not be responsible for the nuclear localization of the protein. The transient complementation system used in this study provides a simple method for functional analysis of essential baculovirus genes in infected cell cultures.

A 302-bp noncoding DNA fragment from the DNA replication module of phage phiSfi21 was shown to protect the Streptococcus thermophilus strain Sfi1 from infection by 17 of 25 phages. The phage-inhibitory DNA possesses two determinants, each of which individually mediated phage resistance. The phage-inhibitory activity was copy number dependent and operates by blocking the accumulation of phage DNA. Furthermore, when cloned on a plasmid, the phiSfi21 DNA acts as an origin of replication driven by phage infection. Protein or proteins in the phiSfi21-infected cells were shown to interact with this phage-inhibitory DNA fragment, forming a retarded protein-DNA complex in gel retardation assays. A model in which phage proteins interact with the inhibitory DNA such that they are no longer available for phage propagation can be used to explain the observed bacteriophage resistance. Genome analysis of phiSfi19, a phage that is insensitive to the inhibitory activity of the phiSfi21-derived DNA, led to the characterisation of a variant putative phage replication origin that differed in 14 of 302 nucleotides from that of phiSfi21. The variant origin was cloned and exhibited an inhibitory activity toward phages that were insensitive to the phiSfi21-derived DNA.

The intracellular localization of the baculovirusAutographa californicanuclear polyhedrosis virusp143gene product (P143) was investigated by immunofluoresence staining of infected and transfected cells. As expected for a protein essential for viral DNA replication, under these conditions, P143 was localized to the nucleus. However, when a plasmid directing the synthesis of P143 from its own promoter was co-transfected with a plasmid expressing IE-1, P143 was found in the cytoplasm. Cotransfection of cells with a series of plasmids expressing viral genes sufficient for stimulation of plasmid replication resulted in nuclear localization of P143 suggesting that one of the gene products required for viral DNA replication may also assist nuclear localization of P143. Sequential deletion of various genes from this assay revealed that when the plasmid expressing LEF-3 was deleted from the mixture, P143 remained in the cytoplasm. Plasmids were constructed where the synthesis of LEF-3 and P143 were directed by theie-1promoter. When these plasmids were cotransfected, both LEF-3 and P143 colocalized to the nucleus suggesting that an important function of LEF-3 is intracellular trafficking of P143 and directing it to the nucleus.

Human cells are generally thought to be nonpermissive for polyomavirus (Py) DNA replication. Using transient transfection, we show that Py large T-antigen (LT) was able to support replication of a Py origin-containing plasmid in two human cell lines. Replication supported by LT in human cells was specific for the Py origin and required its enhancer sequences, as well as the previously reported critical phosphorylation sites within LT. Py replication efficiency was comparable to that of papillomavirus E1- and E2-activated DNA replication in transient assays performed in human 293 and C-33A cells. Previous analysis of DNA replication in vitro has pointed to polymerase α–primase as a specificity determinant for polyomavirus. The data presented here imply that in certain cellular environments, Py LT must functionally interact with human polymerase α–primase to permit DNA replication.

The herpes simplex virus type 1 (HSV-1) helicase-primase, an essential component of the viral DNA replication machinery, is a trimeric complex of the virus-coded UL5, UL8, and UL52 proteins. An assembly of the UL5 and UL52 subunits retains both enzymic activities, and the UL8 protein has been implicated in modulating these functions, facilitating efficient nuclear uptake of the complex and interacting with other viral DNA replication proteins. To further our understanding of UL8, we have constructed plasmids expressing mutant proteins, truncated at their N- or C-termini or lacking amino acids internally, under the control of the human cytomegalovirus major immediate-early promoter. Deletion of 23 amino acids from the N-terminus or 33 from the C-terminus abolished the ability of UL8 to support DNA replication in transient transfection assays. None of the UL8 mutants tested exhibited a strong dominant negative phenotype in the presence of the wild-type product, although some inhibition of replication was observed with mutants lacking 165 N-terminal or 497 C-terminal amino acids. The ability of the UL8 mutants to facilitate efficient nuclear localization of UL52 in the presence of coexpressed UL5 was examined by immunofluorescence. Selected mutants were also expressed by recombinant baculoviruses and tested for interaction with UL5 and UL52 in immunoprecipitation assays. The replicative ability of the mutants was found to correlate with their ability to localize UL52 to the nucleus, but not their interaction with UL5 and UL52. This property precluded the identification of any region of UL8 important for its presumed nuclear functions.

Three viral proteins, all products of early region 2 (E2), participate directly in adenovirus DNA replication. Three products of early region 4 (E4) also affect viral DNA synthesis: the product of E4 ORF4 inhibits viral DNA accumulation, while the products of E4 ORFs 3 and 6 antagonize that effect of ORF4 expression. Because no E4 products are required for DNA synthesis, these proteins probably act indirectly. The E4 ORF3, 4, and 6 proteins all participate in aspects of the regulation of viral gene expression. To determine whether they modulate DNA replication by effects on expression of viral replication proteins, we examined E2 expression in E4 mutant-infected cells. In cells infected by ORF3−, 6−mutants, expression of ORF4 substantially depressed the steady-state levels of replication proteins and E2 mRNAs, reduced E2 transcription rates, and profoundly inhibited viral DNA replication. Thus, in the absence of E4 ORFs 3 and 6, ORF4 acts as a transcriptional regulator of E2 expression, and reduced replication protein levels largely account for the inhibition of DNA replication by ORF4. Cells infected by viruses that express ORFs 3 and 6 in addition to ORF4 accumulated much larger quantities of viral DNA than did cells infected by the ORF3−, 6−, 4+mutant. Increased DNA accumulation was not accompanied by a comparable increase in E2 expression. Therefore, the ORF3 and 6 products counteract the ORF4-induced reduction of DNA replication by a mechanism other than reversing the inhibitory effect of ORF4 on E2 expression. The effect of ORF4 on E2 expression is consistent with its ability to regulate levels of the transcription factor AP-1 (Mülleret al.,1992,J. Virol.66, 5867–5878); the mechanism by which ORFs 3 and 6 enhance replication is unknown.

DNA replication during human or simian cytomegalovirus (CMV) infection has been shown to be under control of a replicator region referred to as ori Lyt. The murine CMV ori Lyt has been mapped to a region of the genome located upstream of the gene encoding the herpesvirus-conserved single-stranded DNA binding protein, analogous to human and simian CMV ori Lyts. A minimal ori Lyt of approximately 1.7 kbp has been identified using a transient replication system. Like occurs with human and simian CMV counterparts, addition of flanking sequences to this minimal origin-stimulated replication efficiency. Analysis of the DNA sequence in this region shows that murine CMV ori Lyt is complex and exhibits an asymmetric distribution of nucleotides as well as many repeat sequence elements, including distinct AT- and GC-rich regions and region with arrays of closely spaced direct repeats. Despite similarities in organization of all three CMV ori Lyts, no sequence identity and only limited DNA sequence similarity was detectable. Consistent with this sequence divergence, the human and murine CMV ori Lyts were unable to substitute for one another in transient replication assays.

In order to define factors involved in very late Autographa californica nucleopolyhedrovirus (AcMNPV) gene function, random mutagenesis of a baculovirus recombinant (AcUW1. lac Z) by 5′-bromodeoxyuridine treatment was performed. Five viruses were selected with deficiencies in very late gene expression. These were characterized by complementation analysis. One mutant virus, VLD1, was found to be completely deficient in very late gene function. This virus could be complemented by a helper virus to express the very late genes, suggesting that the mutant virus was defective in an activator of very late gene expression. Further studies revealed that the replication of VLD1 was temporally delayed when compared to wild-type virus. The mutation in VLD1 was mapped to a subfragment of the Eco RI-I region of the AcMNPV genome between 0 and 5 map units. Sequence analysis revealed the presence of point mutations in ORF2 and in lef -2. Further mapping experiments demonstrated that only replacement of the point mutation in lef -2 with a wild-type sequence could restore VLD1 to a normal phenotype. Previous studies have suggested that the lef -2 gene product is involved in DNA replication. This was investigated by comparison of DNA replication in wild-type- and VLD1-infected cells. It was found that the mutation in the lef -2 gene of VLD1 did not have an effect on DNA replication. It is proposed that lef -2 may play a dual role, both in DNA replication and very late gene expression.

Replication of simian virus 40 (SV40) DNA occurs in SV40 nonpermissive hamster cells upon infection with herpes simplex virus (HSV), leading to concatemeric replication products characteristic for HSV DNA replication. This SV40 origin (ori)-dependent process is governed by SV40 large T antigen and HSV-encoded DNA replication factors; e.g., DNA polymerase, single-strand binding protein (SSB), and helicase–primase. In this study, we show that specific interaction of SV40 T antigen with SV40 ori is crucial for HSV-directed SV40 DNA synthesis and that the property of T antigen to bind and unwind the ori is not sufficient for this process. A T antigen with the mutation T217S, affecting a hypothetical novel DNA replication subfunction, is able to support DNA synthesisin vitrobut not in cultured primate cells. This subfunction is also necessary in HSV-infected hamster cells. Using temperature-sensitive mutants, we demonstrate that the T antigen acts at early stages of DNA synthesis while HSV helicase is required continuously as has been shown for HSV DNA polymerase. HSV SSB is also continuously involved in heterologous SV40 DNA synthesis. However, a HSV mutant, temperature-sensitive in SSB function, showed residual synthesis of SV40 DNA but not of HSV DNA at the nonpermissive temperature. The nature of this dichotomy between HSV SSB function on SV40 DNA and HSV DNA will be discussed.

Herpes simplex virus (HSV) has been shown to induce DNA amplification in the host cell genome, which can be suppressedby the adeno-associated virus type 2 (AAV-2) rep gene (Heilbronn et al. , 1990, J. Virol. 64, 3012–3018). In an attempt to define domains of Rep which are required for this effect a set of expression constructs was generated for Rep mutants with either N-terminal and/or C-terminal truncations, with small internal deletions, or with point mutations. In transient cotransfection assays these mutants were tested for the inhibition of HSV-induced DNA amplification and in parallel for DNA replication of a rep -defective AAV genome. Our data show that the C-terminal region of Rep where spliced and unspliced proteins differ is dispensable for both AAV DNA replication and inhibition of HSV-induced DNA amplification. The N-terminus of Rep is required for AAV DNA replication, whereas the first 174 amino acids can be deleted without loss of function for the inhibition of DNA amplification. Rep52 which starts at methionine 225 is neither sufficient, nor required for this effect. We further analyzed the region between amino acids 174 and 225: A stretch of 16 highly hydrophilic amino acids is dispensable for the inhibition of DNA amplification, but it is required for AAV DNA replication. Deletion of two short motifs spanning putative protein kinase C phosphorylation sites each strongly reduce both AAV DNA replication and inhibition of DNA amplification, whereas a single amino acid substitution of one of these sites abolished AAV DNA replication with no effect on the inhibition of DNA amplification. Our data show that most, but not all, of the sequence elements within the N-terminus of Rep78 required for AAV DNA replication coincide with those required for the inhibition of HSV-induced DNA amplification. A replication-negative version of Rep78 comprising the internal 60% of the protein still carry the entire inhibitory function for HSV-induced DNA amplification.

Intact clones containing partial repeats of the genomic components of African cassava mosaic virus (ACMV DNAs A and B) are infectious when mechanically coinoculated onto Nicotiana benthamiana. Monomeric genomic components may be generated either by homologous recombination or, when two copies of the origin of replication (ori) are present, by a modified rolling circle replication mechanism in which nascent single-stranded DNA is resolved by the introduction of nicks at both oris. DNA B partial repeats with duplicated common region sequences containing combinations of wild-type sequences and nonlethal mutations at nucleotides 151 and 155 within the putative stem—loop region have been constructed and introduced into plants in the presence of DNA A. Analysis of progeny indicates that monomers are generated by DNA strand nicking preferentially between nucleotides 151 and 155, suggesting a nonrandom replicative release mechanism involving the ubiquitous TAATATTAC motif (nucleotides 146–154). Viable ACMV DNA A deletion mutants are known to revert to wild-type size during systemic infection by generating tandem repeats. The recombination point in one such revertant has been mapped between nucleotides 152 and 153. Just as ori-nicking enzymes mediate recombinational events during prokaryotic rolling circle DNA replication, the result suggests that a nick has been introduced in the virion-sense strand within the nonanucleotide motif (TAATATT↓AC) during the initiation of ACMV DNA replication.

Using transient assays, the cis and trans requirements for human papillomavirus type 31b (HPV-31b) replication in transformed and primary keratinocytes have been determined. We demonstrate that expression of both the E1 and E2 open reading frames are necessary and sufficient for replication of a plasmid construct containing a genomic 291-bp fragment in both cell types. The roles of the E1 and E2 proteins in replication were further examined by overexpressing the gene products in a glutathione-S-transferase bacterial expression system. In addition to a full-length E1 protein, a truncated E1 protein (E1*) which consisted of the amino-terminal 268 as was synthesized. Both E1 and E1* were found to complex efficiently with the E2 protein in vivo and in vitro. In addition, site-specific DNA binding by both the E2 and E1 proteins to HPV-31b origin containing DNA was observed. The E1* protein, however, failed to bind HPV-31b DNA specifically and could not cooperate with E2 for replication in transient assays. DNase I footprinting demonstrated that the E1 protein of HPV-31b bound to an A/T-rich region (nucleotides 7905-24) which shares similarities with the binding site for the bovine papillomavirus type 1 (BPV-1) E1 protein. These studies describe both similarities and differences in the requirements for replication of HRV-31b and BPV-1.

The vaccinia virus conditional-lethal temperature-sensitive (ts) mutant ts 4149 is, at the nonpermissive temperature, severely impaired in its ability to replicate its DNA genome. Compared to wild type, the amount of replication is suppressed by several orders of magnitude, and the little DNA that is replicated is not converted to mature linear genomes. We have demonstrated that this "DNA- "phenotype is not the result of a failure to produce early proteins. In agreement with the DNA- phenotype, intermediate and late gene expression were not detected. Marker rescue and DNA sequencing located the mutation in ts4149 to open reading frame D4. This gene has recently been shown to encode a 25-kDa protein with uracil DNA glycosylase activity (D. T. Stuart, C. Upton, M. A. Higman, E.G. Niles, and G. McFadden (1993), J. Virol. 67, 2503-2512). We speculate on the function of this "essential" viral repair enzyme and its role(s) in viral DNA replication.

Human cytomegalovirus transient lytic DNA replication relies on the cis-acting element oriLyt, six viral-encoded core proteins, the proposed DNA replication initiator protein UL84, IE2, IRS1 and the gene products from the UL112/113 loci. In an effort to elucidate cellular and viral-encoded factors that may play a role in oriLyt-dependent replication we used DNA-affinity purification and mass spectrometry to isolate and identify several previously unknown cellular and viral factors that interact with HCMV oriLyt DNA. These proteins include the multifunctional hnRNP-K, BUB3, HMGB1, PTB-1, UL83, UL112/113, and IRS1. Chromatin immunoprecipitation (ChIP) assays confirmed an interaction of several of these factors with oriLyt. Co-immunoprecipitation experiments detected an interaction between UL84 and hnRNP-K in infected and transfected cells. Knockdown of hnRNP K expression by siRNA inhibited the amplification of oriLyt in the transient assay. Together, these data suggest a possible regulatory role in DNA replication for several previously unidentified viral and cellular factors.

Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

The NS-1 gene of the parvovirus minute virus of mice encodes a multifunctional protein essential for viral DNA replication and gene expression. In addition to possessing DNA helicase and ATPase activities, NS-1 forms a covalent linkage with the 5′ ends of viral DNA and is a strong candidate for the site-specific nicking-closing enzyme postulated to be involved in the resolution of concatemers and terminal hairpin structures that arise during parvoviral DNA replication. Since the covalent linkage between NS-1 and the 5′ terminus of MVM DNA resists alkali and mild acid treatment, a tyrosine phosphodiester is likely to be involved. To map domains responsible for this activity, mutations converting tyrosine to phenylalanine were introduced into the NS-1 gene using oligonucleotide-directed mutagenesis and their effect on the DNA replication and transcriptional activation functions of NS-1 was examined in transient in vivo transfection assays. Replacement of Tyr-188, Tyr-197, Tyr-210, Tyr-310, Tyr-422, or Tyr-550 with phenylalanine greatly reduced the ability of NS-1 to complement the replication of the target genome ins 20B in COS-7 cells. However, a Ser-545 to Thr-545 substitution in the Phe-550 mutant restored DNA replication activity. Replacement of 5 other tyrosines in NS-1 with phenylalanine either enhanced (Phe-6), had a moderate inhibitory effect (Phe-209) or had no effect (Phe-47, Phe 227 and Phe-543) on its DNA replication activity. Two of the 11 phenylalanine substitution mutations, Phe-188 and Phe-197, also greatly reduced the ability of NS-1 to transactivate the p38 promoter and displayed a dominant negative phenotype with respect to transactivation. Since the remaining tyrosines in MVM NS-1, Tyr-152, Tyr-252, Tyr-374, and Tyr-595, are not conserved among the NS-1 proteins encoded by porcine and feline parvoviruses, they are presumed to be nonessential for the normal functioning of NS-1. The results point to a role for either Tyr-188, Tyr-197, Tyr-210, Tyr-310, or Tyr-422 in forming a covalent linkage with viral DNA and further suggest a regulatory role for several tyrosines in other DNA replication and transcriptional activation functions of NS-1.

The origin binding protein (OBP) of herpes simplex virus (HSV) type 1 specifically interacts with two high-affinity sites in each HSV DNA replication origin. The sequence-specific DNA binding activity of OBP maps to the carboxy-terminal one-third of the protein. For a single binding site, recombinantly expressed forms of this DNA binding domain have the same sequence specificity and binding affinity as the full-length OBP. However, unlike the full-length protein, truncated OBP does not bind HSV replication origins in a cooperative manner. To determine if cooperative interactions between DNA-bound OBP molecules are essential for viral DNA replication, the 317-amino-acid carboxy-terminal DNA binding domain of OBP was expressed in chick embryo fibroblasts. Cells were infected with HSV type 1, and viral DNA synthesis and virus production were monitored. We found that cells expressing truncated OBP were severely restricted for virus replication and that HSV DNA synthesis was undetectable. The results demonstrate that the amino-terminal two-thirds of OBP is essential for HSV DNA replication and that the OBP DNA binding domain acts as a transdominant inhibitor of viral DNA replication. The results also suggest that this experimental approach could be used to generate a refined map of essential OBP functions and that the approach may be generally applicable to the analysis of the multifunction HSV DNA replication complex.

Replication of the bovine papillomavirus (BPV-1) DNA requires both the viral E1 and E2 gene products. The minimal origin of replication, which resides in a 60-basepair fragment centered on the unique Hpal site of the BPV-1 genome, can be bound by the E1 protein and is flanked by E2 binding sites. The integrity of the region surrounding the unique Hpal restriction enzyme site is important for E1 binding and DNA replication, but the E2 binding sites are not required. The ability of E1 to complex with E2 and the requirement for both factors in DNA replication have led to the hypothesis that the E1/E2 complex may have a direct role in replication. Therefore we have studied mutations in the BPV-1 origin region for E1, E2, and E1/E2 complex binding and for their effect on transformation and replication in the context of the complete viral genome. Transformation and DNA replication were observed with some mutated vital genomes dramatically reduced for E1 binding. These mutated origins could bind significant amounts of E1 in the presence of E2, suggesting that binding of the E1/E2 complex could compensate for the loss of E1 binding in DNA replication. One mutation, with an eight-nucleotide insertion at the Hpa l site, was still bound by E1 and the E1/E2 complex, yet did not replicate or transform efficiently. Thus although the binding of E1 or the E1/E2 complex to the origin appears to be necessary for replication, it is not sufficient.

The papillomavirus life cycle is regulated by a family of proteins encoded by the E2 open reading frame; E2 proteins regulate viral gene expression, DNA replication and genome maintenance. We have previously shown that the bovine papillomavirus (BPV1) full-length E2 protein forms heterodimers with repressor forms of E2, and these E2 heterodimers serve as activators of transcription and replication during the viral life cycle. In the present study, using the single-chain E2 heterodimer as a model, we show that human papillomavirus (HPV) 11 and 18 E2 heterodimers with single activation domain are able to initiate replication of URR-containing plasmid in transient assay. Single-chain E2 heterodimer in the context of HPV18 genome initiates genome replication, but is not sufficient for long-term replication of HPV18 genome. We also show that HPV18 genome has a capacity to encode truncated E2 repressor E8/E2 which acts as a negative regulator of HPV18 genome replication.

Using a combination of Bal31 deletion mutagenesis and site-directed mutagenesis, we analyzed the sequence requirements for the DNA replication of the human neurotropic polyomavirus JC. In addition to defining the late side boundary of the viral core origin we demonstrated that the viral enhancer stimulates replication in vivo. Three regions within the viral enhancer increased the rate of replication, with sequences directly adjacent to the late side of the core origin exhibiting the strongest effect. These sequences interact with various cellular proteins, among them NF-I. Point mutations within the NF-I site abolished the stimulation of DNA replication concomitant with a strong reduction in NF-I binding. By contrast, point mutations which did not interfere with NF-I binding did not influence the rate of replication in vivo. Stimulation of JCV DNA replication could only be observed in vivo, but not in vitro, indicating a role of NF-I in determining chromatin structure.

The papillomaviruses are small DNA viruses that encode approximately eight genes, and require the host cell DNA replication machinery for their viral DNA replication. Thus papillomaviruses have evolved strategies to induce host cell DNA synthesis balanced with strategies to protect the cell from unscheduled replication. While the papillomavirus E1 and E2 genes are directly involved in viral replication by binding to and unwinding the origin of replication, the E6 and E7 proteins have auxillary functions that promote proliferation. As a consequence of disrupting the normal checkpoints that regulate cell cycle entry and progression, the E6 and E7 proteins play a key role in the oncogenic properties of human papillomaviruses with a high risk of causing anogenital cancers (HR HPVs). As a consequence, E6 and E7 of HR HPVs are invariably expressed in cervical cancers. This article will focus on the E6 protein and its numerous activities including inactivating p53, blocking apoptosis, activating telomerase, disrupting cell adhesion, polarity and epithelial differentiation, altering transcription and reducing immune recognition.

The C-terminal 322 amino acids of the varicella-zoster virus (VZV) gene 51 product were expressed in Escherichia coli and shown to bind to specific DNA sequences within the VZV origin of DNA replication. The gene 51 product and its herpes simplex virus type 1 homolog (the UL9 protein) are capable of recognizing identical DNA sequences but the arrangement of binding sites within the origin regions of the two viruses differs. Three binding sites for the VZV gene 51 protein were identified within the VZV origin region and these lie in the same orientation and on the same side of the origin palindromic DNA sequence. DNA replication assays in transfected tissue culture cells demonstrated that the site closest to the palindrome is essential for origin activity whereas the most distal site is dispensable. The middle binding site may play an auxiliary role in DNA synthesis.

Herpes simplex virus induces the activation of the cellular DNA double strand break response pathway dependent upon initiation of viral DNA replication. The MRN complex, consisting of Mre11, Rad50 and Nbs1, is an essential component of the DNA double strand break response and other reports have documented its presence at sites of viral DNA replication, interaction with ICP8 and its contribution to efficient viral DNA replication. During our characterization of the DSB response following infection of normal human fibroblasts and telomerase-immortalized keratinocytes, we observed the loss of Mre11 protein at late times following infection. The loss was not dependent upon ICP0, the proteasome or lysosomal protease activity. Like activation of the DSB response pathway, Mre11 loss was prevented under conditions which inhibited viral DNA replication. Analysis of a series of mutant viruses with defects in cleavage and packaging (UL6, UL15, UL17, UL25, UL28, UL32) of viral DNA or in the maturational protease (UL26) failed to identify a viral gene product necessary for Mre11 loss. Inactivation of ATM, a key effector kinase in the DNA double strand break response, had no effect on Mre11 loss and only a moderate effect on HSV yield. Finally, treatment of uninfected cells with the topoisomerase I inhibitor camptothecin, to induce generation of free DNA ends, also resulted in Mre11 loss. These results suggest that Mre11 loss following infection is caused by the generation of free DNA ends during or following viral DNA replication.

Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

Regulation of DNA repair mechanisms during the viral replication cycle may have consequences for the virus with regards to genomic variability, adaptation, and replication of viral DNA. We have studied the activities and expression patterns of key enzymes involved in the first two steps of base excision repair (BER) of DNA in primary fibroblasts infected by human cytomegalovirus (HCMV). Infected cells were very proficient for removal of uracil and 5' hydrolysis of AP sites (AP endonuclease activity) as compared to the mock-infected cells, suggesting a direct role in generating free ends at uracil lesions in DNA for initiation of viral replication. Furthermore, the capacity to initiate repair of alkylated and oxidized base lesions were reduced in HCMV-infected cells, indicating increased mutation frequencies that could promote genetic variability. We hypothesize that modulation of BER activities may play an important role in HCMV pathogenesis to ensure efficient replication and genomic variation of viral DNA.

Four copies of a hexanucleotide (H) sequence are located to the right of the palindrome at the origin of DNA replication of the porcine circovirus type 1 (PCV1) genome. These sequences are organized in two direct tandems, the proximal H1/H2 and the distal H3/H4 repeats, and they have been shown to be binding sites for the essential Rep and Rep′ proteins. Previous work demonstrated that infectious PCV1 virion can accommodate a variable number of H sequences at the origin of DNA replication. In this work, mutational analysis was conducted to elucidate the critical core element within the hexanucleotide with respect to self-DNA replication and progeny virus synthesis. It was found that while a single H sequence abutting the palindrome is sufficient for PCV1 viability, a tandem repeat arrangement is the more stable and thus preferred configuration. Within the H sequence, selected nucleotides at specific positions are critical for Rep-associated protein recognition and for viral DNA replication.

Human papillomaviruses (HPVs) replicate as nuclear plasmids in infected cells. Since the DNA replication machinery is generally conserved between humans and Saccharomyces cerevisiae, we studied whether HPV-1 DNA can replicate in yeast. Plasmids containing a selectable marker (with or without a yeast centromere) and either the full-length HPV-1 genome or various regions of the viral long control region (LCR) and the 3′ end of the L1 gene were introduced into S. cerevisiae and their ability to replicate episomally was investigated. Our results show that HPV-1 sequences promote episomal replication of plasmids although the yeast centromere is required for plasmid retention. We have mapped the autonomously replicating sequence activity of HPV-1 DNA to a 450 base-pair sequence (HPV-1 nt 6783–7232) that includes 293 nucleotides from the 5′ region of the viral LCR and 157 nucleotides from the 3′ end of the L1 gene. The HPV-1 ARS does not include the binding sites for the viral E1 and E2 proteins, and these proteins are dispensable for replication in S. cerevisiae.

A plasmid-based transfection system capable of generating infectious porcine circovirus type 2 (PCV2) was established. This system was then used in mutagenesis studies to investigate the involvement of a “conserved” nonanucleotide (which constitutes a portion of the loop sequence) located at the origin of DNA replication of PCV2 with respect to viral protein synthesis, DNA self-replication, and progeny virus production. The results demonstrated that an octanucleotide (AGTATTAC) embedded in the loop sequence is essential for virus replication. This octanucleotide can be further condensed to an essential core element (ECE) represented by AxTAxTAC. The positions specified by the indicated nucleotides are critical for viral DNA replication and stable infectious virus production, and they cannot be substituted by other bases, while the positions indicated by x can accept variable bases and yield stable progeny viruses.

During porcine circovirus (PCV) replication in PK15 cells, nine PCV type 2 (PCV2)-specific RNAs are synthesized. They include the capsid RNA (CR), five Rep-associated RNAs (Rep, Rep′, Rep3a, Rep3b, and Rep3c), and three NS-associated RNAs (NS515, NS672, and NS0). In this work, mutational analyses were conducted to investigate the involvement of each PCV2 transcription unit in viral protein synthesis and DNA replication. The results demonstrated that a stop codon introduced at the very 5′-end of CR did not affect Rep-associated antigens or viral DNA synthesis. Altering the consensus dinucleotides at the splice junctions of the minor RNAs (Rep3a, Rep3b, Rep3c, NS515, and NS672) or introducing a stop codon in the abundant NS0 RNA also did not have any effect on viral protein synthesis or DNA replication. However, mutations that resulted in truncated Rep or Rep′ proteins caused greater than 99% reduction of viral protein synthesis and complete shut down of viral DNA replication. These results demonstrated that both Rep and Rep′ are absolutely essential for PCV2 replication.