1. Introduction
BK polyomavirus (BKPyV) is one of more than 10 human
polyomaviruses (
HPyV) and it infects >90% of the general human population without ill effects [
1,
2,
3].
BKPyV causes nephropathy or haemorrhagic cystitis in immunocompromised patients, particularly after kidney or allogeneic hematopoietic cell transplantation [
4,
5]. Seroprevalence data indicate that
BKPyV transmission occurs in early childhood [
6] via the oral or respiratory route [
7]. Subsequently,
BKPyV reaches the renourinary tract, presumably by primary viremia [
7], where the virus establishes a latent infection [
8]. Asymptomatic low-level
BKPyV shedding has been demonstrated in 10% of
BKPyV IgG-seropositive healthy blood donors [
9] providing evidence of immune escape in adult immunocompetent hosts. Antibody levels decline during adult life [
9,
10] unless significant re-exposure occurs which includes immunocompromised patients [
11,
12,
13,
14]. In immunocompromised individuals, however, urinary
BKPyV shedding is more frequent, often with urine viral loads of exceeding 7 log10 copies/mL that become apparent as “decoy cell” shedding [
2,
15,
16,
17,
18]. In kidney transplant or allogeneic hematopoietic cell transplant patients, high-level
BKPyV replication precedes nephropathy or haemorrhagic cystitis, respectively [
4,
5]. Increased
BKPyV reactivation rates have also been observed in other solid organ transplant recipients and in
HIV/AIDS patients [
19,
20,
21]. However, the molecular details of regulating
BKPyV replication in immunocompetent individuals and the relevance for disease progression are not well understood.
BKPyV has a double-stranded DNA genome of approximately 5 kb, which can be divided into: (i) the early viral gene region (
EVGR) encoding the small and the large T-antigen; (ii) the late viral gene region (
LVGR) encoding the capsid proteins Vp1, Vp2, Vp3, agnoprotein and microRNAs; and (iii) the
non-coding control region (
NCCR) [
2,
22]. The
NCCR harbours the origin of genome replication
ori and promoter/enhancer with DNA-binding sites for transcription factors mediating the secondary host cell specificity [
23], as well as the timing and course of
EVGR-expression, viral DNA-replication and
LVGR-expression [
2].
BKPyV sequences commonly found in the urine of healthy persons have an archetype
NCCR architecture of sequence blocks arbitrarily denoted O-P-Q-R-S [
2,
9]. In immunocompromised patients with
BKPyV-disease, viral variants with rearranged
NCCR (rr-NCCR) architecture have been shown to emerge as majority species and are associated with disease severity [
24]. In these patients, archetype
NCCR-BKPyV is still found in urine but molecular cloning has demonstrated the presence of a viral
quasi-species with co-existing
rr-NCCR minority species [
25], which may be an indicator of imminent pathology [
26]. In vitro studies and in vivo observations support the view that
rr-NCCRs confer a higher replicative activity in vitro but which depends on the lack of cellular immune functions in vivo [
24]. A similar link between
rr-NCCR and disease was also observed for
JCPyV in
HIV/AIDS patients with progressive multifocal leukoencephalopathy [
27,
28], or for
HPyV-7 and
HPyV-9 [
23]. Together, the data suggest that
HPyV-NCCR rearrangements arising in immunocompromised patients are not only a surrogate marker of long-standing immunologically uncontrolled replication but also represent a virulence determinant of activated
EVGR expression and increased replication capacity causing disease.
Given the complex diversity of
NCCR rearrangements that affect various transcription factor binding sites as well as the overall architecture, a systematic study of inactivating specific transcription factor binding sites by point mutation was conducted, which maintained the linear archetype architecture of
BKPyV-NCCR [
29]. Of note, mutations inactivating the
Sp1 site located proximal to the
LVGR promoter termed
SP1-4 resulted in a phenotype functionally equivalent to
NCCR rearrangements (group 1) and which had been identified in patients with
BKPyV disease [
29]. Intriguingly, a similar, albeit low-affinity Sp1 binding site
SP1-2 has been located upstream of the EVGR promoter, the inactivation of which (e.g.,
sp1-2) decreased EVGR expression and replication (group 3) [
29]. Further mutational dissection of the
BKPyV-NCCR as well as electrophoretic mobility shift assays and chromatin immunoprecipitation analysis revealed that EVGR-expression involves a classic inducible TATA-box promoter. Upstream, the
EVGR promoter partially overlaps with a constitutive housekeeping gene-type
LVGR promoter using a TATA-like box in the opposite orientation, in which a high-affinity
SP1-4 sites acts as a central switch of bidirectional gene expression [
30].
sp1-4 point mutations inactivating Sp1 binding cause constitutive activation of
EVGR expression and increased viral replication without NCCR rearrangements [
30].
Besides the
NCCR, another layer of regulating EVGR expression has been described at the posttranscriptional level mediated by microRNAs (miRNAs) miRNA-B1-3p and miRNA-B1-5p encoded in the distal
LVGR [
22,
31,
32,
33]. Similar to other
PyVs [
31,
33,
34,
35],
BKPyV miRNAs are short noncoding RNAs, which target large T-antigen transcripts and thereby down-regulate viral replication [
32,
36,
37]. This posttranscriptional safeguard has been implicated in the escape from large T-antigen-specific cytotoxic T-cells [
34], which have recently been linked to the effective curtailing of
BKPyV replication in kidney transplant patients [
38,
39]. The regulation of miRNAs expression is subject of ongoing studies, which may involve sequences close to the miRNA gene as well as the
NCCR [
22,
40,
41]. Moreover, miRNAs of
BKPyV and the closely related
JCPyV miRNAs have been detected in blood, urine and cerebrospinal fluid samples, often together with the corresponding viral loads with few cases reporting the nature of the
NCCR structures [
42,
43]. It has been suggested that urinary exosomes associated
BKPyV-miRNA may be a surrogate marker for
BKPyV pathology [
44]. Thus, the association between miRNAs and exosomes has also raised questions about their regulatory potential in non-infected neighbouring cells [
43,
45,
46].
Since the NCCR and the miRNA represent two different, formally independent modalities of regulating BKPyV replication at the transcriptional and post-transcriptional level, respectively, we examined the interplay of NCCR architecture and miRNAs expression following BKPyV infection. Imperiale and colleagues reported that BKPyV miRNA levels were low following infection with laboratory strains carrying rr-NCCR. In a first step, we therefore compared miRNA levels in archetype and different laboratory- and patient-derived rr-NCCR-BKPyV variants in cell culture. We also examined the miRNA levels seen with the (sp1-4)NCCR-BKPyV and (sp1-2)NCCR-BKPyV, which, as outlined above, have an archetype architecture but show increased and decreased EVGR expression and viral replication, respectively. The role of Sp1 was further evaluated by siRNA-SP1 known-down. To investigate whether or not rr-NCCR-BKPyV replication could still be down-regulated by mircoRNA, we transferred exosomes carrying BKPyV-microRNA cargo onto uninfected cells prior to infection. Finally, we explore the potential relationship of BKPyV load, NCCR architecture and miRNA levels in vivo in urines from multiple sclerosis patients treated with natalizumab. We then attempt to integrate the results in a dynamic model of transcriptional and posttranscriptional regulation and discuss the potential implications in immunocompetent and immunosuppressed hosts, favouring archetype and rearranged NCCR-BKPyV, respectively.
2. Materials and Methods
2.1. Urine Sampling
The urine samples used in this study were obtained from 12 relapsing-remitting multiple sclerosis patients undergoing intravenous natalizumab treatment at the Multiple Sclerosis Center, Neurological Institute Mondino, Pavia, and had enrolled for a previous study [
47]. None of them developed symptoms or signs of polyomavirus-associated diseases at presentation or during the follow-up. The study was approved by the local institutional review board, “Fondazione Istituto Neurologico Mondino”, Pavia, Italy (n. 101MS326). Human samples were taken after obtaining the informed consent from the patients or control subjects in accordance with the tenets of the Declaration of Helsinki.
2.2. Cell Cultures and BkPyV Molecular Clones
African green monkey kidney, SV40-transformed Cos-7 cells line (CRL1651, ATCC, Manassas, VA, USA [
48]) were grown in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS, SIGMA, Milan, Italy). Primary human renal proximal tubule epithelial cells (RPTECs, PCS-400-010, ATCC, Manassas, VA, USA) were grown in an epithelial cell medium supplemented with epithelial cell growth supplement and 2% FBS (ScienceCell Research Laboratory, Carlsbad, CA, USA).
BKPyV strains were derived from molecular clones carrying archetype
NCCR (clone
WW1.4) [
24], a
rr-NCCR laboratory strain (Dunlop, Akron, OH, USA) [
49], clinical rr-NCCR variants having deletions in the Q- and the R-block (clone
del5.3,
del15.10) [
24,
29] and the archetype
NCCR variants carrying point mutations of Sp1 binding sites in the Q-block (clone
sp1-4) or in the P-block (clone
sp1-2) [
29,
30].
2.3. BKPyV Infection of Cos-7 and RPTECs
Transfection of
BKPyV genomic DNA into Cos-7 cells was performed at 90–95% confluence in 6-well plates using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. At 6 h posttransfection, the medium was replaced by DMEM containing 10% FBS. At 14 days posttransfection, Cos-7 cells were harvested by scraping off cells in 1/10 of the cell culture supernatant. Virus was released by 3 cycles of freeze-thawing of the cells and centrifugation at 800
g for 5 min. For the infection experiments, Cos-7 and RPTECs were seeded at 7.5 × 10
5 cells and 3.12 × 10
5 cells per well in 6-well plate with 2 mL of DMEM (10% FBS) or supplemented EpiCM medium (2% FBS). After 24 h at a confluence of approximately 70%, Cos-7 and RPTECs were exposed to 500 microliters of the corresponding virus preparations (MOI 1) obtained from Cos-7 cells after transfection at 37 °C for 2 h followed by removal and replacement with DMEM (10% FBS) or supplemented EpiCM medium (0.5% FBS). Cells and supernatant were harvested at 12, 24, 48 and 72 h post infection. Titration revealed that approximately 5–10 × 10
6 genome copies of
BKPyV-Dunlop genomes on 50,000 RPTECs or Cos-7 cells at infection typically elicits a multiplicity of infection of 1 after 48 h according to fluorescent focus forming units using staining for large T-antigen; H.H. Hirsch and M. Wernli, unpublished results and see References [
50,
51].
2.4. Plasmid Exosomes BKPyV miRNA Expression in Cos-7 Cells
BKPyV-miRNAs were inserted into the pcDNA 6.2GW/EmGFP-miR (Invitrogen, Carlsbad, CA, USA) vectors and cloned according to the manufacturer’s instructions. To this end, specific oligonucleotide pairs were designed on the BKPyV miRNA mature sequence (5′TGCTGATCTGAGACTTGGGAAGAGCATTTTTGGCCACTGACTGAATGCTCTTCCCATCTCAGAT3′ forward oligonucleotide and 5′CCTGATCTGAGATGGGAAGAGCATTCAGTCAGTGGCCAAAAATGCTCTTCCCAAGTCTCAGATC3′ reverse oligonucleotide; BKPyV-miRNA mature sequence is underlined below the sequences shown). Next, the corresponding expression vectors pcDNA6-BK-miRNA (10 µg) were transfected into Cos-7 cells (7.5 × 105 cells per well in 6-well plate) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). BKPyV-miRNA expression vectors system (encodes for the green fluorescence protein GFP) efficiency was assessed at 24h after transfection based on the number of GFP-positive cells, as determined by flow cytometry. In subsequently experiments exosomes carrying BKPyV-miRNA were purified at 24 h, 48 h and 72 h from transfected Cos-7 cells and were quantified. The sequence of the miRNA inserts was confirmed by sequencing.
2.5. Exosome Enriched Vesicles Extraction
The exosome-enriched vesicles were isolated starting from 250 µL of cells supernatant collected by prior centrifugation at 14,000
g for 20 min, using the exosome-specific extraction kit (Norgen, Thorold, ON, Canada), following the manufacturer’s protocols. Characterization of the exosome-preparation was done by demonstrating the presence of 112 nm vesicles present at a density of a mean total particles concentration of 10
7/mL. Further characterization was achieved by western blot, detecting three markers of exosome tetraspanin/CD63, CD81 and Annexin II (
Supplement Figure S1). Before analysis, exosome-enriched vesicles were treated with RNase and DNase to remove miRNA and DNA not protected inside the exosomes.
2.6. Exosome Addition and Anti BKPyV-miRNA Inhibition in Cos-7 Cells
Exosomes carrying BKPyV-miRNA (103 copies) were added to the Cos-7 cells (7.5 × 105 cells per well in 6-well plate) and used to test the effect on subsequent viral infection. Where indicated, the exosomes were added to Cos-7 cells in the absence or presence of inhibitory specific antago-BK-miRNA-5p (Inh-BK-miRNA, a phosphorothioate oligonucleotide with the sequence complementary to the BK-miRNA) synthetic molecule, which had been transfected into Cos-7 cells using a 5 µM final concentration, together with lipofectamine 2000 (Life Technologies, Foster City, CA, USA). Unrelated miRNA (Unrel-miRNA, a phosphorothioate oligonucleotide with an unrelated sequence) synthetic molecule, was used as control.
2.7. BKPyV DNA Quantification
Viral DNA was extracted from 0.15 mL of urine, from 2 × 10
4 cells and cell supernatant of tissue cultures with the High Pure PCR Template Preparation kit (Roche, Basel, Switzerland). DNA extracted was subjected to quantitative real-time PCR (qPCR) assays using primer and probe targeting Vp1 gene (BKVPf forward primer 5′-AGTGGATGGGCAGCCTATGTA-3′, BKVPr reverse primer 5′-TCATATCTGGGTCCCCTGGA-3 and BKVPp TaqMan MGB probe labelled with VIC VIC-5′ AGGTAGAAGAGGTTAGGGTGTTTGATGGCACAG 3′) (Life Technologies, Foster City, CA, USA). Each reaction was carried out with negative controls (no template) and DNA standards (diluted to contain 10
1–10
6 copies per millilitre) of a plasmid containing the
BKPyV molecular clone. The lowest limit of detection of the assay was 10 copies per millilitre of urine and micrograms of total DNA [
52].
2.8. BKPyV Pre-miRNA and Mature miRNA Quantification
Total RNA was isolated from 2.0 × 106 Cos-7 and RPTECs cells using the mirVana miRNA isolation Kit (Ambion, Foster City, CA, USA), and from exosomes contained in 250 µL of cell-free supernatant that had previously been centrifuged at 14,000 g for 20 min using an RNA exosome-specific circulating extraction kit (Norgen). The miRNA expression was measured and quantified with a specific pre-miRNA and mature bk-miRNA-5p quantitative stem-loop RT-PCR MiRNA assay whose primers were designed on the specific region of the BKPyV WW clone (Life Technologies, Foster City, CA, USA) according to the manufacturer’s protocol. Each reaction was performed in triplicate using 10 ng of extracted RNA, including negative controls (no template) and synthesized oligonucleotides as standards (diluted to contain 101–106 copies). The lowest detection limit of the assay was 10 copies/ng of RNA. The assay was specific and reproducible, as demonstrated in preliminary experiments using a BKPyV oligonucleotide standard (with <0.5 Ct value inter-assay variation) and observing no amplification of unrelated oligonucleotide targets.
2.9. siRNA knock-Down of Sp1 and Immunoblotting
Cos-7 cells were transfected in a 6-well plate using per well 2 µg of siRNA targeting Sp1 (SP1-siRNA, Mission esiRNA SIGMA), or 2 µg of siRNA scrambled form (Scr-siRNA), or mock-treated without siRNA added (mock). At 24 h post-transfection, Cos-7 cells were infected at multiplicity of infection (MOI) of 1, using BKPyV-infectious supernatant obtained after transfection of Cos-7 cells. BK-miRNA-5p expression in cells and presence in exosomes enriched vesicles were measured at 48 h post infection. Immunoblots were used to compare Sp1 expression and cytochrome P450 as control. Briefly, cell extracts were prepared from Cos-7 cells at 48 h after transfection of SP1-siRNA, Scr-siRNA and mock-treatment and 15 µg of extract were analysed by 10% SDS-PAGE, transferred onto nitrocellulose membrane (BIO-RAD), block the membrane with 5% bovine serum albumin and then incubated with the rabbit anti-Sp1 (PLA0044, SIGMA) and rabbit anti-cytochrome-P450 polyclonal antibody (PA1-343, SIGMA) followed by a peroxidase-conjugated anti-rabbit IgG antibody (A0545, SIGMA).
2.10. NCCR Sequencing
A nested-PCR was used to obtain the NCCR product for sequencing. Briefly, 100 ng of total DNA was amplified using two pairs of primers: first pair of primers, BKTT1 forward 5′ AAG GTC CAT GAG CTC CAT GGA TTC TTC C 3′ and BKTT2 reverse 5′ CTA GGT CCC CCA AAA GTG CTA GAG CAG C 3′, generating a 684 bp DNA fragment; the second pair of primers, BK-1 forward 5′ GGCCTCAGAAAAAGCTTCCACACCCTTACTACTTGA 3′ and BK-2 reverse 5′ CTTGTCGTGACAGCTGGCGCAGAA 3′, that amplified a portion of the first amplicon generating a fragment of 354 bp. The PCR products were purified using the PCR purification Kit (Qiagen, Hilden, Germany) and sequenced using the BigDye Terminator Cycle-Sequencing Ready Reaction (Applied Biosystems, Foster City, CA, USA). The sequences were analysed and edited using Bioedit 5.0.9 (Tom Hall of Ibis Therapeutics, Carlsbad, CA, USA).
2.11. Statistical Tests
The data were analysed using Student’s t-tests. p-values less than 0.05 were considered statistically significant.
4. Discussion
The regulation of
BKPyV persistence and reactivation is receiving increasing attention [
8] in the light of the almost universal but well controlled
BKPyV infection of the general human population and the increasing
BKPyV diseases in patients under potent immunosuppressive regimens [
4,
5,
54,
55]. So far, two major mechanisms of regulating
BKPyV replication have emerged, which involve transcriptional,
NCCR-based and post-transcriptional miRNA-based mechanisms targeting EVGR expression [
24,
41]. Pioneering work from Imperiale and co-workers has demonstrated that both layers of regulation appear to interact since low
BKPyV miRNA-5p levels were seen in infection by rapidly replicating
BKPyV carrying
rr-NCCRs, whereas high miRNA-5p levels were seen in archetype
BKPyV [
41].
In this study, we extend these observations from laboratory strains to patient-derived isolates and show that
BKPyV miRNA-5p levels are decreased following infection of patient
BKPyV carrying diverse
NCCR rearrangements (
Figure 1). Moreover, we demonstrate that point mutations inactivating a single Sp1 binding site (
sp1-4) in an otherwise archetype
NCCR have a similar effect. This (
sp1-4)-mutation has been shown to confer a phenotype of increased EVGR expression and more rapid viral replication (group 1) similar to
rr-NCCR BKPyV variants [
29]. Thus, our results indicate that NCCR rearrangements are not required for the lowered miRNA levels but suggest that the activation of EVGR expression is important. The latter was supported by another Sp1 point mutant (
sp1-2) showing which permits only modest EVGR expression, while LVGR expression is also reduced (group 3) [
29] and which showed high-levels of BKPyV miRNA similar to the archetype
WW(
1.4)-
NCCR-BKPyV. Of note, the
BKPyV miRNA-5p expression of archetype and (
sp1-2)-mutated
NCCR-BKPyV could be significantly diminished by reduced Sp1 levels following siRNA-
SP1 knock-down. Conversely, variants carrying rearranged or (
sp1-4)-mutated
NCCRs were not affected. Parallel experiments in human RPTECs generated similar results, suggesting that the observations were also relevant in the human situation. The data support the notion that
BKPyV miRNA-5p levels and the
NCCR activity are critically linked by Sp1 binding to the
SP1-4 site. Independently, a principle role of Sp1 for
BKPyV replication has recently emerged from a genome-wide interference study [
56].
While our study provides a detailed accounting of the inverse association of EVGR activity and miRNA-5p expression, the exact molecular mechanisms need to be addressed further. Inverting the orientation of the
rr-NCCR has been shown to confer high LVGR expression at the expense of EVGR [
23,
24] and corresponding recombinant viruses show increased miRNA expression [
41]. Whether such LVGR transcription opens the access to the miRNA promoter region, provides transcription factors and enzymatic complexes, or extended transcripts is presently discussed. Conversely, activated EVGR transcription might simply outcompete the available miRNA transcripts, or confer resistance to downregulation by another as yet unknown mechanism including antisense stealth transcripts or processing from pre-miRNA to miRNA. In our study, we observed that
BKPyV pre-miRNA were similarly affected by the
NCCR activity as the mature miRNA-5p suggesting that transcript generation rather than miRNA processing, maturation or degradation was affected. No evidence for a difference in cellular miRNA by packaging and secretion as exosomes was obtained, since both cellular and exosomal levels mirror-imaged the activity of the NCCR in both compartments. The possibility that
BKPyV miRNAs were no longer able to downregulate replication of variants with activated EVGR was refuted in time course experiments adding
BKPyV-miRNA-5p loaded exosome preparations to host cells prior to infection, which demonstrated a significant reduction of the
BKPyV-Dunlop and the (
sp1-4)-point mutant. This interpretation was supported by inclusion of an antagonist synthetic phosphorothioate oligonucleotide reversing replication inhibition.
The polyomavirus miRNA-5p has been proposed to act as an important safeguard silencing residual large-T-antigen expression of the archetype
BKPyV during viral persistence [
34,
41]. Efficient transcriptional and posttranscriptional synergy in downregulation of large T-antigen expression may permit escape from cytotoxic CD8 T-cell effectors, which we have been shown to preferentially target T-antigen epitopes [
38,
39]. Conversely, a bi-directional link between
NCCR activity and miRNA-expression appears biologically plausible, when signals of activating EVGR expression are sensed in the latently infected host cell, for example by displacing Sp1 from the
SP1-4 binding site in the LVGR promoter [
30]. Downregulating the posttranscriptional miRNA then permits for an efficient progression through the viral life cycle. In immunosuppressed patients lacking sufficient CD8 T-cell activity, the high and prolonged viral replication allows for the emergence of
rr-NCCR variants conferring an activated EVGR expression and high-replication capacity [
24], while reducing posttranscriptional interference through BK-miRNA-5p downregulation
Given the potential clinical relevance for immunocompromised patients, we explored
BKPyV shedding,
NCCR architecture and miRNA levels in multiple sclerosis patients treated with natalizumab. We found that patients shedding
BKPyV with
rr-NCCRs had on average higher urine viral loads but lower miRNA-5p levels in the exosome-enriched vesicles. Conversely, patients shedding
BKPyV with the archetype
NCCR architecture showed lower viral loads and typically higher miRNA-5p levels in urinary exosome preparations. Thus, these preliminary data, if confirmed, seem to be consistent in their almost dichotomous nature and also provide an incentive for further work, which may be of relevance for clinical diagnostic and therapeutic approaches [
42,
44].
Finally, there is increasing evidence reported that other viruses including those that have the propensity to establish latent/persistent infections such as herpesviruses use miRNA regulation not only intracellularly but also in exosomes [
57]. Thus, besides a principle role in virus biology, our data, together with those of other researchers, strongly suggest that viral miRNA should be explored further in a virological and clinical context. To integrate our results into the work of other researchers and to stimulate the corresponding projects, we present a model (see
Figure 8) in which host cell signals, viral
NCCR activity and miRNA expression permit fine tuning of persistence, reactivation of replication, spread to neighbouring host cells, for example, in the epithelial monolayer of the renal tubules and hiding from cytotoxic large T-antigen-specific CD8 T-cells, unless the host is immunocompromised. The potential role of exosomes and miRNA cargo in cell to cell communication offers an interesting possibility that could potentially be harnessed for antiviral therapy.