Kaposi's sarcoma-associated herpesvirus lytic origin (ori-Lyt)-dependent DNA replication: identification of the ori-Lyt and association of K8 bZip protein with the origin

Herpesviruses utilize different origins of replication during lytic versus latent infection. Latent DNA replication depends on host cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin binding protein (OBP) that recruits the core replication machinery. In this report, we demonstrated that DNA sequences in two noncoding regions of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome, between open reading frames (ORFs) K4.2 and K5 and between K12 and ORF71, are able to serve as origins for lytic cycle-specific DNA replication. The two ori-Lyt domains share an almost identical 1,153-bp sequence and a 600-bp downstream GC-rich repeat sequence, and the 1.7-kb DNA sequences are sufficient to act as a cis signal for replication. We also showed that an AT-palindromic sequence in the ori-Lyt domain is essential for the DNA replication. In addition, a virally encoded bZip protein, namely K8, was found to bind to a DNA sequence within the ori-Lyt by using a DNA binding site selection assay. The binding of K8 to this region was confirmed in cells by using a chromatin immunoprecipitation method. Further analysis revealed that K8 binds to an extended region, and the entire region is 100% conserved between two KSHV ori-Lyt's. K8 protein displays significant similarity to the Zta protein of Epstein-Barr virus (EBV), which is a known OBP of EBV. This notion, together with the ability of K8 to bind to the KSHV ori-Lyt, suggests that K8 may function as an OBP in KSHV.     

New insights into replication origin characteristics in metazoans

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins.     

Interaction of Kaposi's sarcoma-associated herpesvirus ORF59 with oriLyt is dependent on binding with K-Rta

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) displays two distinct life stages, latency and lytic reactivation. Progression through the lytic cycle and replication of the viral genome constitute an essential step toward the production of infectious virus and human disease. KSHV K-RTA has been shown to be the major transactivator required for the initiation of lytic reactivation. In the transient-cotransfection replication assay, K-Rta is the only noncore protein required for DNA synthesis. K-Rta was shown to interact with both C/EBPα binding motifs and the R response elements (RRE) within oriLyt. It is postulated that K-Rta acts in part to facilitate the recruitment of replication factors to oriLyt. In order to define the role of K-Rta in the initiation of lytic DNA synthesis, we show an interaction with ORF59, the DNA polymerase processivity factor (PF), one of the eight virally encoded proteins necessary for origin-dependent DNA replication. Using the chromatin immunoprecipitation (ChIP) assay, both K-Rta and ORF59 interact with the RRE and C/EBPα binding motifs within oriLyt in cells harboring the KSHV bacterial artificial chromosome (BAC). A transient-transfection ChIP assay demonstrated that the interaction of ORF59 with oriLyt is dependent on binding with K-Rta and that ORF59 fails to bind to oriLyt in the absence of K-Rta. Also, using the cotransfection replication assay, overexpression of the interaction domain of K-Rta with ORF59 has a dominant negative effect on oriLyt amplification, suggesting that the interaction of K-Rta with ORF59 is essential for DNA synthesis and supporting the hypothesis that K-Rta facilitates the formation of a replication complex at oriLyt.     

Leucine zipper domain is required for Kaposi sarcoma-associated herpesvirus (KSHV) K-bZIP protein to interact with histone deacetylase and is important for KSHV replication

The Kaposi sarcoma-associated herpesvirus (KSHV; or human herpesvirus-8)-encoded protein called K-bZIP (also named K8) was found to be multifunctional. In this study, we discovered that K-bZIP interacts with histone deacetylase (HDAC) 1/2 in 12-O-tetradecanoylphorbol-13-acetate-stimulated BCBL-1 lymphocyte cells. K-bZIP appears to repress HDAC activity through this interaction, which we determined to be independent of K-bZIP SUMOylation. We dissected the domains of K-bZIP and found that the leucine zipper (LZ) domain is essential for the interaction of K-bZIP and HDAC. In addition, we constructed a KSHV bacterial artificial chromosome (BAC) with LZ domain-deleted K-bZIP (KSHVdLZ) and transfected this mutated KSHV BAC DNA into HEK 293T cells. As a result, it was consistently found that K-bZIP without its LZ domain failed to interact with HDAC2. We also showed that the interaction between K-bZIP and HDAC is necessary for the inhibition of the lytic gene promoters (ORF50 and OriLyt) of KSHV by K-bZIP. Furthermore, we found that the LZ domain is also important for the interaction of K-bZIP with the promoters of ORF50 and OriLyt. Most interestingly, although it was found to have suppressive effects on the promoters of ORF50 and OriLyt, KSHVdLZ replicates at a significantly lower level than its BAC-derived revertant (KSHVdLZRev) or KSHVWT (BAC36) in HEK 293T cells. The defectiveness of KSHVdLZ replication can be partially rescued by siRNA against HDAC2. Our results suggest that the function of K-bZIP interaction with HDAC is two-layered. 1) K-bZIP inhibits HDAC activity generally so that KSHVdLZ replicates at a lower level than does KSHVWT. 2) K-bZIP can recruit HDAC to the promoters of OriLyt and ORF50 through interaction with HDAC for K-bZIP to have a temporary repressive effect on the two promoters.     

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA-DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA-DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.     

The functional role of Cdc6 in S-G2/M in mammalian cells

The Cdc6 protein is required for licensing of replication origins before the onset of DNA replication in eukaryotic cells. Here, we examined whether Cdc6 has other roles in mammalian cell-cycle progression from S to G2/M phase. Using RNA interference, we showed that depletion of Cdc6 in synchronous G1 cells blocks G1 to S transition, confirming the essential role of Cdc6 in the initiation of DNA replication. In contrast, depletion of Cdc6 in synchronous S-phase cells slowed DNA replication and led to mitotic lethality. The Cdc6-depleted S-phase cells showed fewer newly fired origins; however, established replication forks remained active, even during chromatin condensation. Despite such DNA replication abnormalities, loss of Cdc6 failed to activate Chk1 kinase. These results show that Cdc6 is not only required for G1 origin licensing, but is also crucial for proper S-phase DNA replication that is essential for DNA segregation during mitosis.     

Evaluation of the lytic origins of replication of Kaposi's sarcoma-associated virus/human herpesvirus 8 in the context of the viral genome

The lytic origins of DNA replication for human herpesvirus 8 (HHV8), oriLyt-L and oriLyt-R, are located between open reading frames K4.2 and K5 and ORF69 and vFLIP, respectively. These lytic origins were elucidated using a transient replication assay. Although this assay is a powerful tool for identifying many herpesvirus lytic origins, it is limited in its ability to evaluate the activity of replication origins in the context of the viral genome. To this end, we investigated the ability of a recombinant HHV8 bacterial artificial chromosome (BAC) to replicate in the absence of oriLyt-R, oriLyt-L, or both oriLyt regions. We generated the HHV8 BAC recombinants (BAC36-DeltaOri-R, BAC36-DeltaOri-L, and BAC36-DeltaOri-RL), which removed one or all of the identified lytic origins. An evaluation of these recombinant BACs revealed that oriLyt-L was sufficient to propagate the viral genome, whereas oriLyt-R alone failed to direct the amplification of viral DNA.     

Kaposi's sarcoma-associated herpesvirus ori-Lyt-dependent DNA replication: involvement of host cellular factors

Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.     

The splicing factor SF2/ASF binds to ARS homologs in a human rDNA replication origin

The function of the relatively well-studied DNA replication origins in the yeast Saccharomyces cerevisiae is dependent upon interactions between origin replication complex (ORC) proteins and several defined origin sequence elements, including the 11 bp ARS consensus sequence (ACS). Although the ORC proteins, as well as numerous other protein components required for DNA replication initiation, are largely conserved between yeast and mammals, DNA sequences within mammalian replication origins are highly variable and sequences homologous to the yeast ACS elements are generally not present. We have previously identified several replication initiation sites within the nontranscribed spacer region of the human ribosomal RNA gene, and found that two highly utilized sites each contain a homologue of the yeast ACS embedded within a DNA unwinding element and a matrix attachment region. Here we examine protein binding within these initiation sites, and demonstrate that these ACS homologues specifically bind the alternate splicing factor SF2/ASF as well as GAPDH in vitro, and present evidence that the SF2/ASF interaction also occurs within the nuclei of intact cells. As the moderate upregulation of SF2/ASF has been linked to oncogenesis through the promotion of alternatively spliced forms of several regulatory proteins, our results suggest an additional mechanism by which SF2/ASF may influence the transformed cell phenotype.     

Cloning, sequencing, and functional analysis of a Marek''s disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.