The TR sequence of the KSHV genome is an essential cis
element for viral replication in latency. The TR functions like an autonomous replicating sequence, and LANA binds to a specific sequence within the TR and supports viral replication (4
). LANA also interacts with many cellular factors, such as RING3 (39
), pRb (43
), p53 (22
), HP1 (37
), CREB-binding protein (36
), mSin3 (35
), MeCP2 (32
), DEK (34
), histone H1 (16
), ORCs (38
), GSK-3β (23
), and Suv39H1 (personal communication). Most of these factors are involved in transcription, chromatin structure, and replication, and some or all of them may be required for viral maintenance as episomes and govern the transcription program in latency. Consistent with this idea, LANA is known to be an essential factor for maintaining the episomal form of the viral genome.
Previous studies indicate that TR and LANA are necessary and sufficient for viral replication and maintenance (3
). The details about this mechanism, however, have been unclear. Unlike the simian virus 40 large T antigen, LANA does not have any enzymatic activity; therefore, during latency, the replication machinery must be recruited to the TR in synchrony with the cell cycle. Although LANA has been shown to interact directly with ORCs 1 to 5 by the glutathione S
-transferase pull-down method (38
), the details about these interactions and their purpose remain unclear. Here we sought to identify cellular factors that associate with TR by generating a TR column and analyzing the factors binding to it. We found that cellular replication machinery components such as CDC6 and MCM7, as well as ORC2, bound to TR in a LANA-dependent manner, which was confirmed by Western blot analysis. These factors are likely to support the viral replication that is dependent on the cell cycle, in cooperation with LANA. ORCs do not appear to recognize any specific nucleotide sequences (52
), and thus, it is probably very important that a DNA-binding protein, such as LANA in this case, lies on the DNA to recruit such DNA replication machinery factors, although the details of this process remain to be elucidated. When we discuss their dependence on LANA, we should be more careful, since we used different cell lines, BC3 (KSHV positive) and BJAB (KSHV negative), whose genetic backgrounds are not totally identical, though both are thought to originate from a B cell lineage. Thus, not only some viral factors but also some cell-specific factors may be required for interaction with LANA.
We also found that some DNA repair components, such as PARP1, MSH2, MSH3, and MSH6, which were identified by MALDI-TOF (MS), interacted with TR. Among these molecules, PARP1 and MSH6 were confirmed to be TR specific and did not bind K-oriLyt CS. PARP1 bound TR independent of LANA, and the other factors may form a complex with PARP1 (42
In this report, we focused on PARP1 because it is reported to be a multifunctional protein and was recently found to act as a component of the centromere and the centrosome (21
), which suggests that it may be a key factor for chromatin structure (51
), replicated genome segregation, and partitioning (50
), although it is best known as a target substrate for the caspases in apoptosis (53
). In KSHV, a recent report showed that PARP1 interacts with RTA, an immediate-early protein that is a key factor for the induction of lytic replication (25
We found that PARP1 bound TR independent of LANA, since the PARP1 in the BJAB lysate also bound TR. Although PARP1 is known to bind double-stranded DNA nonspecifically, under the conditions we used, its binding was specific for TR, given that PARP1 did not bind to K-oriLyt CS. Two 5′-TCGNT-3′ motifs, which are present in the regulatory region of the CXCL1 (41
) gene, are also in TR. Furthermore, PARP1 is reported to be located in the centromere and the neocentromere and to bind to a 5′-GTGAAAAAG-3′ motif in the human centromeric α-satellite DNA and a tandemly repeated AT28 sequence in a cloned region of neocentromere DNA (21
). These sequences are completely different from each other, yet PARP1 was shown to bind to each of them in a specific manner.
Thus, the PARP1-binding sequence may be quite degenerate. We found that one of the two 5′-TCGNT-3′ motifs in the TR may have a higher specificity for PARP1 than the other. This probably means that PARP1's binding to DNA is affected by other factors, such as the flanking sequences and/or the presence of binding proteins such as LANA. In addition, the colocalization of PARP1 and LANA in the infected cells further supported the idea that they interact in vivo and suggested that LANA may be a target of poly(ADP-ribosyl)ation; however, their physical interaction was not proven, as is also the case for PARP1 and p53 (30
). An incorporation assay using biotinylated NAD revealed that LANA was a target of PARP1, and it is likely that the function of LANA is modulated by this modification. HU treatment, which increases PARP1 activity, led to a decrease in the viral genome copy number per cell without affecting cell growth, suggesting that the viral replication and partitioning functions are dependent on the ribosylation status of LANA, although the details remain to be elucidated. On the other hand, NA or 3-AB treatment, which causes a decrease in PARP activity, led to an increase in the viral genome copy number. Thus, the PARP1 activity modulated the viral copy number per cell, and therefore, the level of poly(ADP-ribosyl)ation activity may be critical for viral genome replication and maintenance. Since PARP1 is involved in the function and structure of centromeres and centrosomes (21
), it is reasonable to propose that the replication and partitioning of the viral genome in latency are affected by PARP1.
PARP1 has also been reported to be involved in EBV genome replication and maintenance (18
). PARP1 binds to the EBV oriP
dyad symmetry region in a sequence-specific manner, given that it did not bind to the Zta responsive element. Another similar enzyme, tankylase, which is a telomere-associated PARP (7
), also binds to EBV oriP
in an EBNA1-dependent manner. EBNA1 is poly(ADP-ribosyl)ated, and drugs affecting PARP activity modulate the EBV copy number (18
Poly(ADP-ribosyl)ation is involved in a variety of physiological and pathophysiological phenomena, such as DNA base excision repair, DNA damage signaling, regulation of genomic instability, and regulation of transcription and proteasomal function (7
). PARP1 is a key enzyme and an important factor regulating the host genome structure, replication, and partitioning (33
). Its main activity is the poly(ADP-ribosyl)ation of proteins, with the poly(ADP-ribosyl)ated proteins usually losing their activity (29
). Although different from the ubiquitin system, poly(ADP-ribosyl)ation may be another protein disabling system. Viruses with an extrachromosomal genome, such as the gammaherpesviruses KSHV and EBV, must utilize cellular components in combination with viral gene products to continue to reside in the host cells. Among these, PARP1 may be used by gammaherpesviruses to modulate this process, and it serves as a potential target for drugs aimed at reducing the levels of these viruses in cells.