Almost 2 decades after the etiology of PVAN was linked to acute BKV replication, the cause of the activation of BKV replication in kidney allografts still remains elusive. Immune suppression is associated with the activation of BKV replication (2
), and stress-related injury, repair, regeneration, and differentiation are also associated (27
), but the responsible mechanisms have not been defined.
We have determined that NFI binds to six NFI sites in the BKV archetype NCCR and stimulates DNA replication. NFI sites in P24–37
(NFI-1) and at the P68
junction (NFI-2), proximal to the core-ori, appear to have a higher affinity for NFI and also stronger stimulatory effects on BKV DNA replication than distal sites. Almost all rearranged viruses contain the P block and P-Q junction region spanning these two NFI sites (30
), supporting the notion that they might be particularly important for efficient viral replication in vivo
. Other NFI sites have been implicated in the early-late transcription switch (NFI-3) (42
), the regulation of viral gene transcription in response to the induction of transforming growth factor β (TGF-β) in kidney allografts (NFI-4) (1
), and the modulation of the hormone-mediated stimulation of BKV replication (NFI-5 and -6) (53
We attempted to distinguish the binding of different NFI isotypes to NFI sites using isotype-specific antibodies. Unfortunately, none of the available isotype-specific antibodies work in EMSAs. As NFI isotypes have an identical DNA binding domain that determines the specificity of binding to consensus sites, we speculate that the different NFI isotypes have similar binding affinities in vitro
. However, the binding activity of NFI isotypes in vivo
is likely affected by their interaction and/or competition with other transcription factors (66
) and is challenging to demonstrate with in vitro
assays using purified proteins.
The topography of NFI sites in the archetype BKV enhancer resembles that of NFI sites in the archetype JCV enhancer, except that JCV lacks the NFI-4 site overlapping the Smad3 site (48
). The NFI site closest to the core origin of JCV also stimulates JCV DNA replication in vivo
). JCV also persistently infects the kidney, and the reactivation of JCV in immunocompromised individuals causes progressive multifocal leukoencephalopathy (PML) (88
). Previous analyses of rearranged JCV enhancers in PML patients also revealed a trend similar to that for rearranged BKV enhancers in PVAN: sequences close to the core origin (A to C for JCV and P and the P-Q junction for BKV), which contain the first two NFI sites (NFI-1 and NFI-2), are usually preserved and duplicated (29
). NFI isotype-specific expression determines the tropism of JCV (55
), but functions for different NFI isotypes in replication have not been identified.
A characterization of the NFI isotype-specific function for BKV DNA replication has been attempted with an in vivo replication system, but the results are complicated by the endogenous expression of different NFI isotypes/splicing variants (data not shown). Using the in vitro monopolymerase assay, we have defined the stimulatory activity of the NFIC/CTF1 isotype, the prototype of the NFI family of transcription factors, on the initiation of BKV DNA replication (). The role of other NFI isotypes in DNA replication will be tested with similar systems as purified NFI isotype proteins and antibodies become available.
As NFI stimulates adenovirus (Ad2/5) DNA replication through the recruitment of the Ad pol-pTP (adenovirus DNA polymerase-preterminal protein) complex to the replication origin (9
) and/or the stabilization of the preinitiation complex (59
), we suggest that similar mechanisms promote BKV DNA replication via the recruitment of BKV Tag and Pol-primase to the replication origin. In support of this, we observed that NFI stimulated the initiation of BKV DNA replication in vitro
only at low concentrations of Pol-primase and NFI, whereas at high concentrations of Pol-primase, no stimulation was observed (), which is reminiscent of the stimulation of adenovirus DNA replication in vitro
by NFI (60
) and which is also consistent with the results of our in vivo
competitive replication assays. Furthermore, NFI forms complexes with BKV Tag and Pol-primase; swine NFI also binds to calf primase and stimulates primase activity in a concentration-dependent manner in biochemical assays (24
). All four NFI isotypes were found to interact with BKV Tag in co-IP assays (D).
These results suggest that the formation of NFI-primase and NFI-Tag complexes is important for the initiation of DNA replication, but elucidating the functions of individual complexes is challenging due to the coexpression of different NFI isotypes/splicing variants. Further studies are required to characterize the nature and functions of these interactions.
Although the NFI sites in the BKV NCCR are not required for BKV DNA replication in the absence of a competitor template, NFI sites in the enhancer stimulate BKV DNA replication when Tag or Pol-primase is limiting. This stimulatory activity might be essential in persistent infections, where BKV replicates at very low levels in kidney tubular epithelial cells when low levels of Tag and Pol-primase are expressed (20
), and for the reactivation of BKV replication. The tubular epithelial cells in normal kidneys are terminally differentiated quiescent cells that divide at a low rate (8
) and express small amounts of Pol-primase (36
). Ubiquitously expressed NFI may facilitate the low-level replication of persistent BKV in kidney epithelial cells by increasing the level (and activity) of Pol-primase at the core-ori. Also, signaling mediated through TGF-β (3
), tumor necrosis factor alpha (TNF-α) (4
), and oxidative stress (5
) induced by kidney ischemia/reperfusion injury and/or inflammatory responses (12
) during kidney transplantation or by the administration of immunosuppressive drugs such as tacrolimus and cyclosporine (67
) might alter NFI isotype expression or activity and thereby promote the NFI-mediated recruitment of Tag and/or Pol-primase to the viral core-ori. These notions can be tested experimentally.