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Presented in part: 13th International Symposium on NeuroVirology, San Diego, California, 3 June 2015.
We document a unique DNA recombination between polyomavirus JC (JC virus [JCV]) and Epstein-Barr virus (EBV) at sequences of JCV found infecting the brain. Archetype JCV is present in bone marrow and uroepithelial cells of most adults. During immunosuppression, JCV can infect the brain, causing a demyelinating disease, progressive multifocal leukoencephalopathy. Rearrangements in the archetype noncoding control region are necessary for neurovirulence. Two NCCR deletions and a duplication occur at sequences of homology with EBV, present latently in B cells, which may be coinfected with both viruses. Recombination between JCV and EBV occurs in B lymphoblasts at a sequence essential for JCV neurovirulence and in cerebrospinal fluid of immunosuppressed patients with multiple sclerosis, those susceptible to progressive multifocal leukoencephalopathy. Interviral recombination is a model for conferring advantages on JCV in the brain. It can alter a critical noncoding control region sequence and potentially facilitate use of EBV DNA abilities to transfer among different cell types.
JC virus (JCV) and Epstein-Barr virus (EBV) are DNA viruses of the polyomavirus and herpesvirus families, respectively. EBV (170–180 kb, linear with latent circular episomes) is nearly ubiquitous in adults, where it is normally latent in B lymphocytes. In addition to infectious mononucleosis, EBV is causally linked to nasopharyngeal carcinoma, and lymphomas, often under conditions of immunosuppression (IS) . A recent report documents that nearly 75% of adults have been infected by JCV although there are age-related variations in this number . JCV (5.1 kb, circular) normally inhabits bone marrow and uroepithelial cells as an archetypal form existing asymptomatically. In AIDS and in persons treated with certain IS agents, however, the virus infects oligodendroglial cells in the brain, causing progressive multifocal leukoencephalopathy (PML), a frequently fatal demyelinating disease [3–5].
The sequence of JCV infecting glial brain cells almost always differs from that of the archetype in the noncoding control region (NCCR), the region containing the origin of DNA replication and separating the early and late gene transcription units. There are 3 NCCR DNA rearrangements that characterize PML: a deletion of approximately 23 base pairs (bp), another deletion of 66 bp and a duplication of 98 bp that remain after the 2 archetype deletions [6, 7]. Although archetype JCV can be detected in some cases of PML, all 3 rearrangements are associated with the worst prognoses . These rearrangements in PML can all be derived from the archetype sequence, and they may do so within a single individual in a sequential order, possibly in different cell types in transit from distal tissues to the brain . In cerebellar granular cell degeneration, distinct from PML, patients frequently have mutations in the JCV VP1 coding region. In several JCV clones from this disease the virus had both 23- and 66-bp NCCR deletions seen in PML but not the duplication [10–12], essentially in agreement with our concept of sequentiality. Because PML does not develop in all persons with these 3 rearrangements, they are considered necessary but not sufficient.
Coinfection of individual B cells by both JCV and EBV has been visualized in a central nervous system lymphoma . JCV reportedly replicates in B cells, including those infected with EBV . B cells reportedly can carry JCV to the brain  even in the absence of persistent replication. JCV can induce DNA double-strand breaks , in cells including B cells . These breaks can initiate recombination . JCV is present in bone marrow [19, 20] as well as in CD19+ and CD34+ B cells in patients with multiple sclerosis (MS) . A regulatory interaction between JCV and EBV in B cells has previously been implied . Here we present evidence that JCV and EBV can undergo a unique interviral recombination that may help facilitate the ability of JCV to adapt to neurovirulence in glial cells. This recombination reveals a mechanism of opportunistic creation of virus variants that represents a new consideration in disease development.
Cell lines used were Raji, a B lymphoma cell type (American Type Culture Collection [ATCC] CCL-86), and SVG cells, astroglia constitutively expressing SV40 large T antigen (ATCC CRL-8621). CD19+ primary B cells were ordered from HemaCare Bioresearch Products. Raji and B cells were cultured in suspension . SVG cells were cultured as described in Supplementary Methods.
DNA recovered from Raji cells was amplified using polymerase chain reaction (PCR), as described in the Supplementary Methods.
Patients' cerebrospinal fluid (CSF) samples were obtained from PrecisionMed. All of the patients were properly consented volunteer donors, and CSF samples were collected using institutional review board–approved protocols. All samples are from the United States and were obtained through several participating physicians. Four available samples were from patients with MS having received treatment with natalizumab alone as the only immunomodulatory or IS agent. Samples from patients and MS stages of patients studied are given in Supplementary Methods, as are purification and amplification methods for CSF DNA.
Raji cells and B cells were subjected to nucleofection, as described in the Supplementary Methods.
Raji cells, primary B cells and SVG cells were fixed and stained as described in the Supplementary Methods . Throughout the study, all data points were repeated at least 3 times. With regard to patient samples, which are difficult to obtain, data from individual patients were repeated.
We used a temperature range of PCR amplifications to reveal the most prominent secondary structure sites of DNA polymerase stalling  (Supplementary Figure 1). These sites in the NCCR sequence are noted by red arrows in Figure Figure1.1. NCCR rearrangements, shown for the Mad-1 strain of neurovirulent JCV, illustrated with an archetype sequence in Figure Figure1,1, include 23-bp and 66-bp deletions. A 98-bp duplication of the sequence remaining after the deletions begins at the first black-lettered c after the origin. An analysis of our own sequencing data, those of several groups, including Reid et al , and of nearly 3000 JCV entries in GenBank has allowed us to draw certain conclusions as to the frequencies of the different NCCR deletions before PML. There are multiple NCCR entries containing one of the PML-related deletions but not the other. Of these, 93% contain the larger, approximately 66-bp deletion, and 7% contain the smaller, approximately 23-bp deletion. Thus the 66-bp deletion is more frequent and probably occurs before the 23-bp deletion.
Because we intended to determine whether JCV DNA recombination can occur in B cells, which may harbor EBV, we used the National Center for Biotechnology Information BLAST algorithm to compare the entire JCV archetype NCCR sequence with the entire wild-type EBV genome (GenBank 507799.2), using a window of 10 nucleotides to look for identities. Only 3 identities longer than that were found (Figure (Figure1),1), containing 15, 14, and 13 consecutive nucleotide identities. All 3 identities are at ends of the 3 major JCV rearrangements (blue arrows in Figure Figure1;1; JCV sequences from GenBank, JO2226.1 [Mad-1] and JX273163.1 ). The 15-bp identity is a complex sequence expected to occur once per 109 nucleotides. The probability of this occurring in the EBV genome is P < 104. The probability that it would occur in both viral genomes is infinitesimally small. The 14-bp identity is at an end of the large T-antigen–binding palindrome and at the precise early region end of the 98-bp duplication. It is a degenerate and repetitive sequence and can be found at several sites in human and herpes virus genomes. The 13-bp identity is at the end of a palindrome marking the early region end of the 66-bp deletion. Again, the probability that this identity would occur in both EBV and JCV is very small. The proximity to palindromes and to elongation stop points (Figure (Figure1)1) renders all identities good candidates for recombination between JCV and EBV initiated by abortive JCV DNA replication. The 15-bp identity is within the 23-bp JCV deletion and is the first sequence information linking this deletion to a potentially specific recombination event.
The 15-bp identity between JCV and EBV is in the EBV gene encoding the protein, BGLF5 (Figure (Figure22A). No alkaline exonuclease of any other herpesvirus possesses the VFEPAP motif (Figure (Figure22A) encoded partially by the 15-bp homology. The 14-bp identity with JCV is in the EBV gene encoding BOLF1, a putative tegument protein (Figure (Figure22B). The 13-bp identity is in the EBV gene encoding LF1, a multifunctional dUTPase (Figure (Figure22C). It is unlikely that recombination events at these sites would be detected in vivo in EBV. There is no foreseeable advantage to EBV for recombination at any of these sites, in which case recombinants would not prevail. In focusing on the 15-bp identity, we can address an important question: can recombination between these 2 viral genomes be detected in a cell harboring both?
To examine recombination at the 15-bp identity, we used Raji cells, a Burkitt lymphoma line harboring EBV. In Raji cells, EBV is latent and nonproductive owing to viral genomic deletions. Raji cells contain 50–80 circular EBV episomes that replicate once per cell cycle using EBV latent oriP and cellular minichromosome maintenance helicase machinery [25, 26]. It has been reported that EBV-containing B cells can support JCV replication . Supplementary Figure 2 shows staining for JCV T antigen in Raji cells, primary CD19+ B cells and positive control SVG glial cells. All have been nucleofected with complete archetype JCV circular DNA. Proteins influencing both JCV and EBV DNA replication are colocalized near the nuclear periphery during times of viral replication . In Supplementary Figure 2F, F’ shows T antigen at the nuclear periphery of B cells. T-antigen binding sites are present throughout human chromosomal DNA , but their distance from the 15-bp homology is most likely much greater than it is in EBV.
To directly assay for the occurrence of recombination at the 15-bp identity, we initially used nucleofected Raji cells. For Figure Figure3,3, a plasmid bearing the complete archetype JCV genome was transfected into Raji cells. At 72 hours, DNA was extracted, and PCR was performed (Figure (Figure33A, Nucl. Arch, nucleofected archetype lane). Several bands were obtained, including a dense one at 177 bp. DNA was similarly analyzed from nonnucleofected cells to which the archetype plasmid was added after lysis. No bands were seen in this mock-archetype lane. The plasmid-only and the no-DNA-template lanes were similarly free of bands. Thus, PCR products containing sequences from both viruses were only obtained when Raji cells were nucleofected with the JCV genome. The band at 177 bp was excised and sequenced using a primer from either JCV or EBV (Figure (Figure33B and and33C). Completely complementary overlapping sequences were obtained using these primers (electropherograms of sequencing in Figure Figure3).3). The 15-bp homology was nearly central in the PCR segment.
The possibility that this result is a PCR artifact is negligible. First, controls in which both viral genomes are present do not yield any recombinant bands. Second, the transition point for recombination occurs exactly at the 15-bp identity. Finally, JCV bears a single-nucleotide polymorphism in the sequence. The strain of JCV used in Figure Figure33 contains the sequence GTAAAAC, whereas EBV contains the sequence GTCAAAC. Sequencing shows that some products begin with GTA, and others with GTC (boxes in Figure Figure3).3). This polymorphism indicates that the products sequenced came from one virus or the other and not because of PCR infidelity. Approximately 0.1% of archetype sequences in GenBank begin the 15-bp homology with GTC, and the others begin with GTA. This single-nucleotide polymorphism helps verify recombination.
Another concern was that our recombination product could be an artifact of overlap-extension synthesis occurring during PCR. We controlled extensively for that. Our mock-archetype lane in Figure Figure33A uses supercoiled plasmid containing JCV DNA. As another control, we have added single-stranded JCV DNA replication intermediates, purified from a standard NCCR-containing PCR reaction, to an EBV-containing B-cell lysate before PCR, using the same primers as for our mock lane. In this case, we do not see any bands containing EBV-JCV recombined sequences. These negative control results are not shown. They strengthen the genomic sequencing data of Figure Figure3,3, suggesting JCV-EBV recombination.
MS is an ideal model to search for disease-relevant recombination because MS is strongly serologically linked to EBV [29, 30], and IS treatment is a known cofactor in causing PML [4, 31]. Although natalizumab is frequently described as an IS agent, its action can be categorized as immunomodulatory. Here we have used IS to denote natalizumab treatment. CSF was obtained from several persons with or without MS and with or without IS treatment. Lanes 1 and 2 of Figure Figure44A reveal no significant EBV bands in persons without MS using PCR with primers for EBV alone. We detected higher EBV levels in patients with MS not treated with IS than in controls without MS. Owing to the small number of subjects, the significance of this difference should be further evaluated. Lanes 3–6 in Figure Figure4,4, from patients with MS treated with IS, show EBV bands of varying intensity. Lanes 7 and 8, from patients with MS not treated with IS, show faint bands. Because MS stages differ between patients, however, we cannot at this time assume any causal relationship among natalizumab treatment, CSF EBV levels, and JCV recombinant formation. None of the patients tested has had PML diagnosed. The band for Raji cells in lane 10 is presented as a control.
Figure Figure44B shows PCR reactions performed on CSF with a forward primer from EBV and a reverse primer from JCV, that, the opposite orientation to that of Figure Figure3.3. The band expected if recombination occurs at the 15-bp homology would be 421 bp. PCR of CSF from a patient without MS shows no potential recombination band. Both patients with MS (corresponding to lanes 3 and 7 in Figure Figure44A) showed bands of the anticipated size. Figure Figure44C shows real-time PCR for the bands of Figure Figure44A and and44B. It can be seen that the relative amount of DNA for the MS + IS3 CSF sample is much higher than those for the other samples. This result is not reflected in Figure Figure44A because by the time bands in 4A are highly visible, the other samples have had several cycles to catch up. Patients without MS have essentially no JCV-EBV recombined DNA in their CSF, whereas the patients with the highest CSF levels of EBV and recombined DNA were those treated with IS, that is, those susceptible to PML (Figure (Figure44C). Again, we cannot presently conclude a causal relationship between natalizumab treatment and JCV-EBV recombinant formation; larger sample sizes would be required to establish any possible causal relationship. Although neither EBV nor recombined DNA is present at high levels in CSF, based on ease of detection by PCR, the levels of unrecombined EBV are much higher than those of the recombined form. The EBV level in the highest MS + IS sample is 860 pg/mL CSF. This is in comparison with a control standard of EBV plasmid DNA (BGLF5-pCDNA3.1). All of the patients' CSF samples were tested for JCV DNA using several sets of PCR primers. We do not detect intact, independent JCV DNA in CSF from any of our patient samples. This indicates that any JCV DNA detected in the CSF is in the form of JCV-EBV recombinants. It is also conceivable that recombination between JCV and EBV occurs in a cell outside the CSF before gaining access to that compartment.
The sequence DNA of the MS + IS3 lane (Figure (Figure44D) is larger than the 421 bp between the primers because each primer was used to sequence separately in reverse order. Sequencing reveals the same JCV-EBV recombination transition as seen in Figure Figure4.4. Although the number of patient samples here is small, the proof of principle is that JCV-EBV recombination can be detected in patients with MS susceptible to PML. More samples will be analyzed to affirm and extend these CSF studies.
We verified recombination between JCV and EBV with a second method using EBV PCR primers only to generate a hybridization template. No JCV primers have been used, and no JCV sequences have been added to the patients' nonprocessed CSF samples. Therefore, if JCV sequences are recombined with EBV, they have not been artifactually derived by our methods. Our EBV primers all bracket the 15-bp homology with JCV in the BGLF5 gene. Agarose gel bands were subjected to Southern blotting using either a phosphorus 32–labeled JCV archetype genome or a labeled EBV BGLF5 fragment. The EBV band from the BGLF5 coding region containing the 15-bp homology, using one of the primer pairs specified in “Methods” section, would be 378 bp. There is no band of this size hybridizing to JCV. Figure Figure55 shows results of hybridization of blots to either JCV or EBV probes. Results show that patients 3–5 (with MS and no IS) have no JCV sequences in bands higher than the expected EBV band, although the patient of lane 5 does harbor EBV. No JCV is associated with any of the EBV bands at 378 bp. In contrast, there are strong JCV bands at the positions of EBV bands of approximately 5 kb and higher. These bands contain both EBV DNA (white arrows) and potentially incorporated JCV DNA (black arrows).
To our knowledge this is the first demonstration that 2 entirely different classes of DNA virus can undergo recombination in a cell harboring both. Although this was demonstrated in a B-lymphoblast line, it may not necessarily occur in B cells in persons, and the possibility that it occurs in bone marrow remains speculative. Estimates of the fraction of B cells that harbor latent EBV in approximately 90% of adults vary from 1 to 50 per 106 cells. EBV infects a variety of epithelial cells [32–34], and JCV infects renal tubular epithelial cells [3, 35]. Epithelial cells, lytically infected with EBVl including nasopharyngeal cells, can fuse  The proximity of these cells to tonsillar cells harboring JCV [31, 37] provides a potential mechanism for transfer of recombinant JCV among cell types. Nanbo and colleagues  have described cell-to-cell contact-mediated EBV transmission.
BGLF5 (mutated in Figures Figures33 and and4)4) is an alkaline nuclease, expressed only during active infection, that shuts off host cell messenger RNA function and would also shut off JCV protein expression. The strategic RNA-binding “bridge” in BGLF5 of gamma herpesviruses is a flexible motif that spans the active sites of alkaline nuclease activity . Mutations in it are especially prone to disrupting host cell shutoff. Because latent EBV has 50–80 episomes, it is unlikely that mutation in this region in one or a few episomes would have any effect on EBV. Its advantage to JCV could be in allowing JCV to gain a foothold in a new cell type after cell-cell transfer .
MS represents an intriguing intersection of EBV, JCV and IS, although neither virus is firmly linked to MS etiology. Natalizumab, an antibody to integrin α4, is associated with many current non-AIDS cases of PML, and PML has developed in a small percentage of patients receiving this treatment [4, 31, 39]. Prior infection with EBV is strongly serologically linked to MS, although the virus is not consistently found in MS lesions . IS activates proliferation of B cells infected with EBV , presumably predisposing any JCV-coinfected cells to opportunistic recombination. All of the NCCR rearrangements described can be derived from interviral recombination among JCV molecules alone , so that recombination with EBV is not necessarily the exclusive route for rearrangement of JCV. What advantage, then, is accrued by JCV through recombination with EBV? Several intriguing possibilities present themselves. Insertion of JCV sequences into EBV at the 15-bp homology can create a duplication of the homology at each end of the insert. That creates a potential fork-stalling palindrome. There is no such fork-stalling site at the 23-bp deleted sequence in JCV alone. Thus recombination with EBV generates a favorable feature promoting JCV recombination. No known single model of recombination can account for the types of recombinants seen in Figures Figures33 and and4.4. Supplementary Figure 3 presents a hypothetical model involving cooperation of several known recombination modes [43, 44].
Figure Figure44 is of key importance to this study because it seems to imply a relationship between natalizumab treatment, CSF EBV levels, and levels of JCV-EBV recombinants. The nuances and caveats of this interpretation are discussed in the Supplementary Notes to Figure Figure44.
Under the right circumstances, EBV can mediate cell fusion and syncytium formation [36, 40, 41]. Glial cells transfected with an EBV-based CD4 expression vector have been observed to undergo fusion . Furthermore, induction of lytic EBV infection of Burkitt lymphoma cells reportedly stimulates cell-to-cell contact-mediated EBV transmission . By capitalizing on such EBV capabilities, JCV DNA may be transported “piggy-back” into cell types not normally infected by JCV. It may then recombine with itself, or as a JCV-EBV hybrid, to advance progression of JCV to neurovirulence.
JCV-EBV recombination is potentially critical because, even if it occurs on a small scale, cells bearing JCV-EBV recombinants can gain advantages regarding transit of JCV into new cell types and enhanced ability of JCV to undergo further recombination capable of generating neurovirulent rearrangements. Interviral recombination involving DNA viruses may be applicable to several viruses in addition to JCV and EBV. This possibility may have implications for the etiology or treatment of several diseases in which interaction of viruses can confer an adaptive advantage on certain of these viruses.
Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.
Acknowledgments.We thank Dr Lindsey Hutt-Fletcher, PhD, of Louisiana State University Health Sciences Center, Shreveport, for review, comments and discussion regarding EBV and Dr H. J. Delecluse, PhD, University of Heidelberg, for generously providing EBV BGLF5 complementary DNA clone, plasmid BGLF5-pCDNA3.1. Malcolm Hall, BA, reviewed the manuscript for scientific clarity.
Financial support.This work was supported by the PML Consortium (E. M. J.) and the National Institutes of Health (D. C. D.).
Conflicts of interest.All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.