Variation in human herpesvirus 6B telomeric integration, excision, and transmission between tissues and individuals

Human herpesviruses 6A and 6B (HHV-6A/6B) are ubiquitous pathogens that persist lifelong in latent form and can cause severe conditions upon reactivation. They are spread by community-acquired infection of free virus (acqHHV6A/6B) and by germline transmission of inherited chromosomally integrated HHV-6A/6B (iciHHV-6A/6B) in telomeres. We exploited a hypervariable region of the HHV-6B genome to investigate the relationship between acquired and inherited virus and revealed predominantly maternal transmission of acqHHV-6B in families. Remarkably, we demonstrate that some copies of acqHHV-6B in saliva from healthy adults gained a telomere, indicative of integration and latency, and that the frequency of viral genome excision from telomeres in iciHHV-6B carriers is surprisingly high and varies between tissues. In addition, newly formed short telomeres generated by partial viral genome release are frequently lengthened, particularly in telomerase-expressing pluripotent cells. Consequently, iciHHV-6B carriers are mosaic for different iciHHV-6B structures, including circular extra-chromosomal forms that have the potential to reactivate. Finally, we show transmission of an HHV-6B strain from an iciHHV-6B mother to her non-iciHHV-6B son. Altogether, we demonstrate that iciHHV-6B can readily transition between telomere-integrated and free virus forms.

been associated with multiple sclerosis and other chronic neurological conditions (Yao et al., 2010). 80 Herpesviruses typically achieve latency by forming circular DNA episomes within specific cell types, 81 but HHV-6A/6B episomes have not been detected and it has been proposed that latency of these 82 viruses is achieved through telomeric integration (Arbuckle et al., 2010). remaining four repeat patterns, two were in iciHHV-6B samples (NWA008 and DER512) that share 158 the same 17p integration site, and two were in acqHHV-6B samples (SAL023 and TEL-FA G1P1). 159 Remarkably, the pvT1 repeat pattern was different in almost every unrelated individual with 160 acqHHV-6B (61/63, 96.8%) and also in the majority of iciHHV-6B individuals (30/39, 77.0%; 29/31, 161 93.5%, when the entire 9q iciHHV-6B group was excluded). 162 Although DR R -pvT1 is highly variable among acqHHV-6B genomes in unrelated individuals, 163 this is not the case within families ( Figure 1C the human genome (Riethman, 2008), the chromosomal locations in these iciHHV-6B clades will 205 require further verification by an independent method, but they represent two more independent 206 germline telomeric integration events. 207 As an aid to analysing the increasing number of HHV-6A/6B genome sequences, we 208 developed HHV-6 Explorer (https://www.hhv6explorer.org/), which is an online interface for 209 monitoring clade-specific variation in DNA and predicted protein sequences. An analysis of sequence 210 variation in relation to integration site using this tool produced new insights, for example by 211 demonstrating that two potentially inactivating mutations, U14 in an iciHHV-6B clade and in U79 in 212 an iciHHV-6A clade, must have arisen after integration (Figure 2 -figure supplement 1). 213 214

Predicting chromosomal iciHHV-6B integration sites from DR R -pvT1 repeat patterns 215
To explore whether DR R -pvT1 repeat patterns reflect the phylogenetic relationships between 216 iciHHV-6B genomes, 19 for which DNA was available, were subjected to DR R -pvT1 analysis. The 217 results show that iciHHV-6B genomes with a high degree of overall sequence similarity also shared 218 similar DR R -pvT1 repeat patterns. For example, individuals with 9q iciHHV-6B have a characteristic 219 CTATGG-TTAGTG-CTATGG motif in the middle region of DR R -pvT1, as well as a TTAGAG repeat that 220 is found infrequently outside this group. Also, viral sequences assigned to the 17p major iciHHV-6B 221 group by genome sequence identity and shared 17p subtelomere-iciHHV-6B junction sequences 222 (HGDP01065, HGDP01077, DER512, 801018 and ORCA1622) (Zhang et al., 2017) had similar DR R -223 pvT1 repeat patterns with two characteristic GTAGTG repeats at the left end ( Figure 3A). 224 From these observations, we hypothesised that DR R -pvT1 repeat patterns may be used to 225 predict both the integration site for newly identified iciHHV-6B carriers and the identity of closely 226 related viral strains, without the need to sequence viral genomes. Based on similarity between DR R -227 pvT1 repeat patterns, the integration site was predicted for nine iciHHV-6B genomes for which 228 genome sequences were not available ( Figure 3A). For example, the repeat patterns in YOR546, CRL-229 1730 and 6-iciHHV-6B were identical or almost identical to those in a large group of iciHHV-6B 230 genomes with a 9q integration site (Nacheva et al., 2008;Shioda et al, 2018). PCR amplification of 231 the subtelomere-iciHHV-6B junction was used to validate several of the predictions arising from 232 these results. For example, DR R -pvT1 analysis placed 410005 and 308006 in the same phylogenetic 233 clade as the 401027 iciHHV-6B genome ( Figure 3, Figure 2), and common ancestry was confirmed by 234 sequence similarity across the subtelomere-iciHHV-6B junction fragment (amplified by primer 2p2, 235 Figure 3B-C, Figure 2) in all three samples. Similarly, the iciHHV-6B genomes in d44, LAT018, 236 NWA008 and KEN071 share DR R -pvT1 repeat patterns and subtelomere-iciHHV-6B junction 237 fragments similar to the large group of iciHHV-6B genomes integrated at 17p (17p major) ( Figure 3B-238 C, (Zhang et al., 2017)). In summary, the integration sites of iciHHV-6B genomes that had not been 239 sequenced were predicted based on a high degree of similarity between DR R -pvT1 repeat patterns, 240 and six of these predictions were subsequently tested and validated by an independent method. 241 242

Chromosomal integration of acqHHV-6B 243
Telomeric integration provides a means by which acqHHV-6A/6B may achieve latency. 244 Although de novo integration has been shown to occur in cell culture ( droplet PCR (ddPCR) assays to quantify HHV-6B genome copy number per cell ( Figure 4A). Using this 249 sensitive approach, low-level HHV-6B was detected in 44 samples (84.6%) with a mean value 250 0.00145 copies per cell (range 0.0000233-0.0125). Two kidney DNA samples, K1 and K10, were also 251 positive for HHV-6B at 0.000525 and 0.0124 copies per cell, respectively ( Figure 4A, D). 252 Previously we used Single TElomere Length Analysis (STELA), a PCR-based method, to detect 253 and measure the length of iciHHV-6A/6B-associated telomeres at DR L -T1 (Huang et al., 2014). To 254 determine whether some copies of acqHHV-6B are integrated into telomeres, we aimed to detect 255 these potentially rare events using STELA. The first step involved STELA of genomic DNA from saliva 256 samples that had low but measurable levels of HHV-6B DNA. The STELA PCR products were then 257 diluted and subjected to a second round of PCR with primers TJ1F and DR421R to amplify DR L -pvT1. 258 Successful amplification in the second round was taken as evidence that an HHV-6B-associated 259 telomere had been amplified in the primary STELA round ( Figure 4B-C). Sanger sequencing was used 260 to confirm DR L -pvT1 amplified from STELA products matched that amplified from genomic DNA from 261 each individual. Various steps were taken to avoid false positive results. These include the use of 262 newly designed STELA primers for these experiments ( Figure 4B) and conducting STELA on genomic 263 DNA from the HT1080 cell line (HHV-6A/6B negative) to check for potential non-specific 264 amplification from another telomere or elsewhere in the human genome. No STELA products were 265 generated from HT1080 among 900 STELA reactions, total 4.5g DNA screened. This two-step STELA 266 procedure, conducted on DNA from six saliva and two kidney samples identified a small number of 267 amplicons from some samples with acqHHV-6B (Supplementary Table 3). The proportion of HHV-6B 268 genomes from which a telomere was amplified was estimated using the HHV-6B copy number per 269 cell (determined by ddPCR) and by estimating the number of cell equivalents of DNA used per STELA 270 reaction, assuming 6.6 pg DNA per cell ( Figure 4D, Supplementary Table 3). For the eight samples 271 analysed, an average of 0.95% (range 0-1.98%) of HHV-6B genomes resulted in pvT1 amplification, 272 indicative of telomeric integration. The low copy number of HHV-6B in these samples, combined 273 with reduced PCR amplification efficiency of longer molecules (telomeres in this case), has a 274 stochastic effect on the potential to detect an HHV-6B-associated telomere in each STELA reaction 275 therefore, the integration frequencies should be interpreted cautiously. 276 We then sought to measure the proportion of acqHHV-6B genomes that are not integrated 277 in saliva samples, using ddPCR to quantify the copy numbers of DR and PAC1. PAC1 and PAC2 are 278 genome packaging signals located at the end of T1 and T2, respectively ( Figure 4B). The linear, 279 unintegrated genome has two copies of DR and two copies of PAC1 and PAC2, whereas the 280 integrated genome has two copies of DR but retains only a single copy of PAC1 and PAC2 ( Figure 4B The histone deacetylase inhibitor, trichostatin-A (TSA) has been shown to promote iciHHV-305 6A reactivation in cell lines and cultured T cells from iciHHV-6A/6B individuals (Arbuckle et al., 2010;306 Arbuckle et al., 2013), and also to increase the abundance of t-circles (Zhang et al, 2019). To explore 307 whether the frequency of truncations with short telomere formation at DR L -T2 could be influenced 308 by the chromatin state of the iciHHV-6B genome, the 4B-11p15.5 lymphoblastoid cell line was 309 treated with TSA. The cells were grown in medium supplemented with various concentrations of TSA 310 for approximately 5 days, and the DR L -T2 truncation assay (DR L -T2-STELA) was conducted on DNA 311 extracted from TSA-treated and control cells ( Figure 5D). The frequency of truncation events at DR L -312 T2 increased significantly from 0.0162 to 0.0290 per cell at the highest TSA concentration. This 313 suggests that iciHHV-6B chromatin conformation influences the chance of t-loop formation at the 314 telomere-like repeats within the viral genome and subsequent excision events. 315 Loss of the terminal DR L via t-loop formation and excision at DR L -T2 was expected to 316 generate a short novel telomere detected as a STELA fragment consisting of a flanking region of DR L 317 of approximately 600 bp plus a telomere repeat array limited to the length of the DR L -T2 ( Figure 5A). 318 Some of the DR L -T2-STELA amplicons were larger than the expected 0.7-0.9 kb ( Figure 5B). We 319 showed that these amplicons lack DR3 or DR8 sequences ( Figure 5E), thus indicating they are not 320 molecules truncated at different sites within DR L . We hypothesized that the intermediate length 321 amplicons (longer than 0.7-0.9kb, Figure 5B) could have arisen by telomerase-mediated lengthening 322 of newly formed telomeres at the DR L -T2 truncation site. To address this, six of the lengthened 323 amplicons from three DNA samples (4B-11p15.5, d56 and NWA008) were sequenced. All comprised 324 the expected flanking sequence followed by a uniform array of (TTAGGG) repeats exceeding the 325 known length of DR L -T2 for the sample, thus showing that some of the novel telomeres formed at 326 DR L -T2 had been lengthened ( reciprocal product of U-DR excision is expected to be a truncation of the iciHHV-6B genome at DR R -340 T1 with new telomere formation. To explore this, we exploited the variable nature of pvT1. 341 Differences between DR R -pvT1 and DR L -pvT1 within a single iciHHV-6B genome were found in many 342 of the iciHHV-6B samples analysed (24/35, 68.6%) ( Figure 6A, Figure 6 -figure supplement 1). These 343 differences were usually a consequence of loss or gain of a small number of repeats, although in 344 some cases they involved single base substitutions that converted one repeat type to another. These 345 differences made it possible to measure the frequency of truncations and new telomere formation 346 at DR R -T1 via a two-step process. 347 First, the iciHHV-6B-associated telomeres were amplified using STELA with the DR1R primer 348 that can generate products from a telomere at DR L -T1 (from normal, full-length iciHHV-6B) or DR R -T1 349 (following U-DR excision). Second, to distinguish between these STELA products, the PCR amplicons 350 were subjected to nested PCR to amplify DR L -pvT1 and DR R -pvT1 ( Figure 6A). The sizes of the pvT1 351 amplicons indicated whether the STELA products in the primary PCR had been derived from 352 telomeres at DR L or DR R ( Figure 6B), and selected sequencing was used to confirm. Truncations at 353 DR R -T1 were detected in all ten iciHHV-6B samples analysed, and the frequency of newly formed 354 telomeres at DR R -T1 was estimated to be 0.0002-0.0029 per cell ( Figure Figure 3B). Using this approach, we found that the DR-only iciHHV-6B in SAL018 and 369 KEN071 share the common 17p (major) subtelomere-iciHHV-6B junction that is also found in 370 801018, d44 and others ( Figure 3C). Similarly, the pattern of telomere and degenerate repeats 371 across a subtelomere-iciHHV-6B junction (amplified by subtelomere primer 2p2) in the DR-only 372 iciHHV-6B sample 308006, closely matched those in full length iciHHV-6B individuals 401027 and 373 410005 ( Figure 3C). The existence of shared integration sites for the full-length and DR-only iciHHV-374 6B carriers at two different chromosome ends clearly establishes that the DR-only status has arisen 375 independently, on at least two occasions, by loss of U and one copy of DR in the germline of an 376 ancestor with a full-length iciHHV-6B genome. suggests that opportunities for reactivation may be more common than currently appreciated. To 385 explore this, we used DR R -pvT1 analysis to investigate the relationship between strains of iciHHV-6B 386 and low-level acqHHV-6B within families. In one family (Rx-F6a), the mother (G3P1) had a low 387 acqHHV-6B load in saliva (0.00035 copies per cell), but both her children were iciHHV-6B carriers. 388 The DR R -pvT1 repeat patterns in the children were identical and presumed to have been inherited 389 from the father, who was not available for testing. Clearly the mother in this family had a different 390 HHV-6B strain with a distinct DR R -pvT1 repeat pattern (Figure 7). 391 Quantification of HHV-6B in saliva samples from another family (Rx-F3a) showed that the 392 mother (G3P2) was an iciHHV-6B carrier and that her daughter (G4P3) had approximately 2 copies of 393 iciHHV-6B per cell. Identical DR R -pvT1 repeat patterns between the mother and daughter proved 394 maternal inheritance of one iciHHV-6B copy. A second, distinct DR R -pvT1 repeat pattern in the 395 daughter was assumed to represent iciHHV-6B inherited from the father, who was not available for 396 testing. The son (G4P1) had 0.00025 copies of HHV-6B per cell in saliva DNA, consistent with the 397 level expected from acqHHV-6B infection. Importantly, the DR R -pvT1 repeat pattern in the son 398 (G4P1) was identical to that in the mother and one copy of iciHHV-6B in his sister. This repeat 399 pattern was not seen outside this family among 102 DR R -pvT1 repeat patterns (Figure 1 -figure  400 supplement 1). These observations strongly suggest that iciHHV-6B genome excision occurred in the 401 mother and that the reactivated HHV-6B was then transmitted to the non-iciHHV-6B son, who 402 retained residual viral sequences in his saliva. shown by the evidence that community-acquired HHV-6 transmission usually occurs between family 443 members, with maternal transmission most common. We showed that DR R -pvT1 repeat patterns can 444 be used effectively to discriminate between HHV-6B strains circulating in communities (61/63 445 different pvT1 sequences in saliva from healthy non-iciHHV-6B donors in the UK). In the future, pvT1 446 analysis could be used to trace patterns of transmission more generally. This may be particularly 447 important in the setting of organ and tissue transplants (Hill, 2019). For example, reactivation of 448 iciHHV-6B from a donor tissue could be monitored and differentiated from HHV-6B acquired by the 449 recipient in early childhood or to identify cases of multiple infections by different strains of HHV-6B. 450 The iciHHV-6B and free HHV-6B viruses in communities must have evolutionary histories 451 that are interlaced but these are difficult to disentangle as there is little understanding of HHV-6 452 telomeric integration and iciHHV-6B excision and reactivation. The current picture indicates a 453 modest number of ancient iciHHV-6B clades (in Europe and North America at least) some of which 454 are accumulating mutations that would prevent full reactivation (discussed above). There is also at 455 least one example of an iciHHV-6B genome with high sequence homology to a group of genome 456 sequences from acqHHV-6B ( has occurred independently at least twice suggests this is a relatively common event, and this is 498 supported by our evidence that new telomere formation at DR R -T1 occurs at a measurable 499 frequency in sperm DNA. Second, we show an example of probable reactivation of iciHHV-6B in a 500 mother with transmission to her son, who carries a very low load of acqHHV-6B with the same 501 distinctive DR R -pvT1 repeat pattern in his saliva. This observation warrants further research to 502 determine how frequently HHV-6B is transmitted from iciHHV-6B parent to their non-iciHHV-6B 503 children but it requires the use of a hypervariable marker, such as pvT1, that has the power to 504 discriminate between HHV-6B strains. The HHV-6 Counter takes a fasta multiple alignment and compares each sequence to a selected 605 reference HHV-6 strain to generate counts of genetic variation (substitutions, insertions, deletions) 606 from overlapping or non-overlapping windows across the HHV-6 genome. If a corresponding 607 genbank file for the reference is present, HHV-6 Counter will also provide windowed counts for 608 coding sequence and amino acid variation across the genome and for each gene. This includes 609 missense/nonsense changes, in-frame insertions/deletions, nonsense insertions, potential splice site 610 changes, loss of start or stop and tracking of frameshift changes. Windows which contain non-611 standard base characters or have sequence gaps due to method failures are flagged but their counts 612 are still retained in the results. HHV-6 Counter exports the count windows both as a series of Excel 613 files and as a python panda pickle file for use in the HHV-6 Explorer. 614 The HHV-6 Explorer is based on plotly Dash (https://dash.plotly.com/) and, using the output from 615 HHV-6 Counter, allows the graphical display of variation counts for different strains across the HHV-6 616 genomes and/or on a per gene basis compared against a selected reference HHV-6 strain. It also 617 displays a multiple alignment for the selected gene. A pre-populated version of HHV-6 Explorer 618 containing the data from this manuscript can be found at https://www.hhv6explorer.org/ . The 619 source code for HHV-6 Explorer and HHV-6 Counter is available from 620 https://github.com/colinveal/HHV6-Explorer. The telomere at the end of the iciHHV-6B genome was amplified by STELA using the DR1R primer 647 and Telorette2/Teltail essentially as described previously (Huang et al., 2014) (Jeyapalan et al, 2008). To detect telomeres at DR R -T1, STELA was conducted as above using primers DR1R and 654 Telorette2/Teltail on iciHHV-6B DNA samples that showed a length difference between DR L -pvT1 and 655 DR R -pvT1. Next the STELA reaction product (1µl) was diluted 1:10 in water and used as input for PCR 656 of pvT1 using primers DR421R and TJ1F (25 cycles). The amplicons were size-separated by agarose 657 gel electrophoresis to distinguish DR R -T1-associated telomeres from the majority of DR L -T1-658 associated telomeres. The remainder of the undiluted STELA product was size-separated by agarose 659 gel electrophoresis and amplified telomeres detected by Southern blot hybridisation to a 660 radiolabelled (TTAGGG) n probe. 661 To detect telomeres at DR L -T2, primer UDL6R was used in STELA reactions instead of primer DR1R 662 with 250-1000 pg genomic DNA per reaction and cycled 26 times. Amplicons hybridising to the 663 radiolabelled (TTAGGG) n probe that migrated at less than 900 bp were counted as unlengthened 664 truncations, those between 900 bp and 8.6 kb were counted as lengthened truncations, and those   DNA and iciHHV-6B carrier status. Father (grey filled square) was not available for testing. Right, the 1202 DR R -pvT1 repeat maps from family members. Daughter (G4P3) has two copies of iciHHV-6B, one 1203 copy shares the same DR R -pvT1 as seen in her mother (G3P2) and a second copy has a different DR R -1204 pvT1 repeat pattern assumed to have been inherited from her father. The son (G4P1) has a very low 1205 level of HHV-6B in his saliva with the same pvT1 repeat pattern as the maternal iciHHV-6B genome. 1206 The DR R -pvT1 repeat patterns are also labeled with m (present in mother); d (present in daughter) 1207 and s (present in son). 1208     The 102 DR R -pvT1 repeat patterns shown demonstrate the diversity among iciHHV-6B (inherited) and HHV-6B (acquired) genomes. Ninety of the repeat patterns were unique. Degenerate, telomere-like repeats present in the HHV-6B pvT1 region are colour-coded: Dark green, TTAGGG; brown, CTAGGG; cyan, TTAGTG; yellow, TTACTG; dark yellow, ATAGAC; teal, CTAAGG; pink, CTATGG; lime green, TTATGG; blue, GTAGTG; peach, TTAGAG; red, GTCTGG. Black squares represent other, less common degenerate repeats and white squares show where the sequence could not be determined accurately. Dashes between repeats were added to maximize alignment between samples allowing comparison between the left, middle (highly variable) and right (highly conserved) sections of DR R -pvT1. Repeat maps were grouped primarily based on the left section, then on less common features within the middle or right region, and finally by the length of the middle region. Ninety eight of the 102 DR R -pvT1 repeat patterns are from unrelated individuals and four are from children who have an acqHHV-6B that has a different DR R -pvT1 repeat pattern from their parents (blue diamonds). Coloured arrows identify 12 repeats patterns found more than once in donors not known to be related. Among eight of the identical DR R -pvT1 repeat patterns (blue arrows), seven are in iciHHV-6B genomes predicted to be integrated in the 9q telomere and one in an acquired HHV-6B in SAL030. Two others, in NWA008 and DER512 from the UK, have a 17p iciHHV-6B integration and share identical DR R -pvT1 repeat patterns. In addition, the acquired HHV-6B strains in SAL023 and TEL-FA G1P1 share the same DR R -pvT1 repeat pattern.

Figure Supplements and Supplementary Tables
Black boxes surround repeat maps from iciHHV-6Bs predicted to have the same integration site, by whole viral genome sequence homology, FISH, or subtelomere-iciHHV-6B junction sequence. Notably within integration groups there are often shared characteristic features in the DR R -pvT1 repeat patterns.
Black dots indicate iciHHV-6B samples where DR R -pvT1 could not be used to predict the integration site confidently. For example, the DR R -pvT1 in iciHHV-6B in d32 has a unique right section and is unlikely to share common ancestry with any of the other iciHHV-6B carriers analysed.
Yellow diamond identifies the DR R -pvT1 repeat pattern in family Rx-F3a, transmitted from iciHHV-6B mother to non-carrier son. This pvT1 repeat map is different from the other 101 maps shown here. HHV-6B strain identification using DR R -pvT1 repeat patterns in eight families, suggests that transmission of acqHHV-6B is predominantly from parents.
The key to the repeat pattern is the same as in Figure 1. Pedigree symbols shaded blue indicate the child(ren) had the same acqHHV-6B DR R -pvT1 repeat pattern as their mother and yellow indicates it was the same as their father. The pedigree symbols shaded grey identify individuals from whom the pvT1 region could not be amplified. The green symbols identify children that do not share a DR R -pvT1 map with either parent and the dark green symbol for TEL-FM G2P1 indicates that this individual had a DR R -pvT1 that was different to their siblings and parents. TEL-FL G2P2 (asterisk) has a DR R -pvT1 repeat pattern that differs from that in their mother by a gain of two repeats (CTAGGG-TTAGGG) in the middle section. The HHV-6 explorer allows different types of variation across iciHHV-6A/6B and acquired HHV-6A/6B genomes from different individuals to be compared against a reference genome. The user can select the size of overlapping or non-overlapping sliding windows in an upper and lower graph to allow easier comparison when a larger number of genomes are selected from the drop-down menus. After selecting the type(s) of variation and genomes to be explored, a lower and upper Genome View graph will be generated based on the user-selected sliding window size. The Genome View shows the specified type(s) of variation across the whole genome as a count of mutations per window size (100 bp, non-overlapping in the examples shown here). Cumulative mutations per window size are shown if multiple types of variation are selected. Each genome is assigned a unique colour and where part of sequence is missing, a value of negative one will be assigned for that particular window and the bar on the graph will be red.
In both examples, truncations (i.e. a nonsense mutation encoding a premature stop codon) are displayed. For HHV-6A, seven 17p iciHHV-6A genomes have been selected to be displayed on the lower graph and a variety of acquired HHV-6A and 18q, 19q and 22q iciHHV-6A genomes have been selected to be displayed on the upper graph. The Genome View is zoomed in to show a region displaying truncations in U79 in three 17p iciHHV-6A genomes (303-046, 303-035 and GLA_15137). In the HHV-6 Explorer the next graph generated is the Gene View where a single gene of interest can be selected to view variation at the amino acid level, U79 in this case. The final plot on the HHV-6 Explorer is the Gene Alignment, based on an alignment of all iciHHV-6A and acquired HHV-6A (or iciHHV-6B and acquired HHV-6B) used to generate the phylogenetic networks. In this region of U79, the sequence assemblies are missing data in 13 of the viral genomes (dashes). The exact amino acid position of the premature stop codon for 303-035 is highlighted, underscore at amino acid 431. All other iciHHV-6A/HHV-6A genomes (for which sequence is available) lack this premature stop codon, including other 17p iciHHV-6A genomes (e.g. 7A-17p13.3) indicating that this mutation arose after integration.
In the right-hand column, members of the iciHHV-6B 17p (minor) integration group are displayed in the upper graphs and a variety of other iciHHV-6B integrations and acquired HHV-6B genomes are displayed on the lower graphs. HST was used as the reference strain here. A premature stop codon is present at position 116 in U14 in 1B-iciHHV-6B but not in 704-021 or 704-016, which share the same common ancestor. Again, this indicates that the nonsense mutation arose after integration.  (A) Schematic of DR-circle assay. DR-circles were amplified using primers (DR8F(A/B) and DR3R) that should not generate a PCR product from full-length iciHHV-6B but should amplify across telomere repeats in a DR-circle, the predicted reciprocal product of a t-loop mediated excision event at DR L -T2. Genomic DNA from two iciHHV-6B individuals (1B-iciHHV-6B and 4B-11p15.5) was digested with combinations of restriction enzymes that cut in the unique region and DR (X: SacI and PstI), only the unique region (Y: XbaI and ScaHF) or were not treated with restriction enzymes. Treated DNA was amplified using DR8F(A/B) and DR3R, and PCR products were size separated and detected by Southern blot hybridized to a radiolabelled (TTAGGG) n telomere probe. Amplicons of variable length were detected reflecting different lengths of telomere repeat arrays expected in individual DR-circles. Restriction digestion with SacI/PstI, including between DR8F(A/B) and T2, prevents amplification from DR-circles. Importantly, the minimal size of amplicons detected was greater than the combined length of the flanking regions. Full length 4B-11p15.5 had longer average telomere length at DR L -T1 than 1B-iciHHV-6B, which is consistent with the longer products generated in the DR-circle assay.
(B) The schematic shows how newly formed telomeres at DR L -T2 were detected and sequenced. UDL6R-STELA was used to amplify telomeres at DR L -T2 and it occasionally amplified telomeres that were longer than the length of DR L -T2. Six of these intermediate length STELA products from three different DNA samples were re-amplified in a semi-nested, secondary PCR using primer DR8RT2 and Telorette2. Following gel extraction these products were Sanger sequenced using DR8RT2 or Telorette2. The TTAGGG repeats were visualized with FinchTV and counted manually. Electropherograms from one reamplified molecule from NWA008 are shown. As expected, the PAC2 motif was present (boxed sequence). A black line at base 273 shows where DR L -T2 in this sample was expected to end (after 18 TTAGGG repeats). Over 100 telomere repeats were counted from Telorette 2, considerably more than the length of T2 showing that this telomere has been lengthened.
(C) Detecting telomerase activity in iciHHV-6B lymphoblastoid cell lines. Telomere repeat amplification protocol (TRAP) was used to detect low levels of telomerase activity in various iciHHV-6B lymphoblastoid cell lines. Water and CHAPS were used as negative controls and cell lysate from a telomerase positive HUV-EC cell line was used as a positive control.   Figure 6 -figure supplement 1. The majority of iciHHV-6B genomes show sequence differences between DR R -pvT1 and DR L -pvT1. DR R -T1 and DR L -T1 were specifically amplified by PCR using DR1R and U100Fw2 for DR R -T1, or by STELA using DR1R and STELA oligonucleotides (Teltail and Telorette 2) for DR L -T1. The products from DR R -T1 and DR L -T1 specific PCRs were used as input for secondary, nested PCR using DR421R and TJ1F to specifically amplify DR R -pvT1 and DR L -pvT1. Sanger sequencing was carried out using the TJ1F primer. Sequences were manually examined to generate pvT1 repeat maps. Repeats maps were colour coded and aligned as in Figure 1 -figure supplement 1. The majority of iciHHV-6B genomes (24/35, 68.6%) showed differences between DR R -pvT1 and DR L -pvT1, usually as loss or gain of a small number of hexameric telomere (TTAGGG) or degenerate telomere-like repeats. In a smaller number of cases, a single base change converted one repeat type to another. Regions containing CTAGGG repeats were most prone to loss or gain of repeats. Where differences between DR R -pvT1 and DR L -pvT1 were detected in multiple iciHHV-6B genomes from the same integration group (boxed) the differences were rarely the same between individuals. This indicates that the differences arose after integration. Interestingly, 9q iciHHV-6B genomes did not display any variation between DR R -pvT1 and DR L -pvT1.