Major Internal Repeat Research Articles

Variation within major internal repeats ofKSHV invivo.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), yet the viral genetic factors that lead to the development of KS in KSHV-infected individuals have not been fully elucidated. Nearly, all previous analyses of KSHV genomic evolution and diversity have excluded the three major internal repeat regions: the two origins of lytic replication, internal repeats 1 and 2 (IR1 and IR2), and the latency-associated nuclear antigen (LANA) repeat domain (LANAr). These regions encode protein domains that are essential to the KSHV infection cycle but have been rarely sequenced due to their extended repetitive nature and high guanine and cytosine (GC) content. The limited data available suggest that their sequences and repeat lengths are more heterogeneous across individuals than in the remainder of the KSHV genome. To assess their diversity, the full-length IR1, IR2, and LANAr sequences, tagged with unique molecular identifiers (UMIs), were obtained by Pacific Biosciences' single-molecule real-time sequencing (SMRT-UMI) from twenty-four tumors and six matching oral swabs from sixteen adults in Uganda with advanced KS. Intra-host single-nucleotide variation involved an average of 0.16 per cent of base positions in the repeat regions compared to a nearly identical average of 0.17 per cent of base positions in the remainder of the genome. Tandem repeat unit (TRU) counts varied by only one from the intra-host consensus in a majority of individuals. Including the TRU indels, the average intra-host pairwise identity was 98.3 per cent for IR1, 99.6 per cent for IR2 and 98.9 per cent for LANAr. More individuals had mismatches and variable TRU counts in IR1 (twelve/sixteen) than in IR2 (two/sixteen). There were no open reading frames in the Kaposin coding sequence inside IR2 in at least fifty-five of ninety-six sequences. In summary, the KSHV major internal repeats, like the rest of the genome in individuals with KS, have low diversity. IR1 was the most variable among the repeats, and no intact Kaposin reading frames were present in IR2 of the majority of genomes sampled.

Intra-host changes in Kaposi sarcoma-associated herpesvirus genomes in Ugandan adults with Kaposi sarcoma.

Intra-host tumor virus variants may influence the pathogenesis and treatment responses of some virally-associated cancers. However, the intra-host variability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma (KS), has to date been explored with sequencing technologies that possibly introduce more errors than that which occurs in the viral population, and these studies have only studied variable regions. Here, full-length KSHV genomes in tumors and/or oral swabs from 9 Ugandan adults with HIV-associated KS were characterized. Furthermore, we used deep, short-read sequencing using duplex unique molecular identifiers (dUMI)-random double-stranded oligonucleotides that barcode individual DNA molecules before library amplification. This allowed suppression of PCR and sequencing errors to ~10-9/base as well as afforded accurate determination of KSHV genome numbers sequenced in each sample. KSHV genomes were assembled de novo, and rearrangements observed were confirmed by PCR and Sanger sequencing. 131-kb KSHV genome sequences, excluding major repeat regions, were successfully obtained from 23 clinical specimens, averaging 2.3x104 reads/base. Strikingly, KSHV genomes were virtually identical within individuals at the point mutational level. The intra-host heterogeneity that was observed was confined to tumor-associated KSHV mutations and genome rearrangements, all impacting protein-coding sequences. Although it is unclear whether these changes were important to tumorigenesis or occurred as a result of genomic instability in tumors, similar changes were observed across individuals. These included inactivation of the K8.1 gene in tumors of 3 individuals and retention of a region around the first major internal repeat (IR1) in all instances of genomic deletions and rearrangements. Notably, the same breakpoint junctions were found in distinct tumors within single individuals, suggesting metastatic spread of rearranged KSHV genomes. These findings define KSHV intra-host heterogeneity in vivo with greater precision than has been possible in the past and suggest the possibility that aberrant KSHV genomes may contribute to aspects of KS tumorigenesis. Furthermore, study of KSHV with use of dUMI provides a proof of concept for utilizing this technique for detailed study of other virus populations in vivo.

Essential role of inverted repeat in Epstein-Barr virus IR-1 in B cell transformation; geographical variation of the viral genome.

Many regions of the Epstein–Barr virus (EBV) genome, repeated and unique sequences, contribute to the geographical variation observed between strains. Here we use a large alignment of curated EBV genome sequences to identify major sites of variation in the genome of type 1 EBV strains; the CAO deletion in latent membrane protein 1 (LMP1) is the most frequent major indel present in the unique regions of EBV strains from various parts of the world. Principal component analysis was used to identify patterns of sequence variation and nucleotide positions in the sequences that can distinguish EBV from some different geographical regions. Viral genome sequence variation also affects interpretation of genetic content; known genes, origins of replication and gene expression control regions explain most of the viral genome but there are still a few sections of unknown function. One of these EBV genome regions contains a large inverted repeat sequence (invR) within the IR-1 major internal repeat array. We deleted this invR sequence and showed that this abolished the ability of the virus to transform human B cells into lymphoblastoid cell lines.This article is part of the theme issue ‘Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses’.

Heterogeneity of the Epstein-Barr Virus (EBV) Major Internal Repeat Reveals Evolutionary Mechanisms of EBV and a Functional Defect in the Prototype EBV Strain B95-8.

ABSTRACTEpstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified through both coevolution with its host and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging because of the large number and lengths of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat 1 of EBV (IR1; also known as the BamW repeats) for more than 70 strains. The diversity of the latency protein EBV nuclear antigen leader protein (EBNA-LP) resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 open reading frame (ORF) is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp) and one zone upstream of and two within BWRF1. IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as from spontaneous mutation, with interstrain recombination being more common in tumor-derived viruses. This genetic exchange often incorporates regions of <1 kb, and allelic gene conversion changes the frequency of small regions within the repeat but not close to the flanks. These observations suggest that IR1—and, by extension, EBV—diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four nonconsensus variants within a single IR1 repeat unit, including a stop codon in the EBNA-LP gene. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 bacterial artificial chromosome (BAC).IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population but causes illness in only a small minority of people. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity to see if different strains have different disease impacts have excluded regions of repeating sequence, as they are more technically challenging. Here we analyze the sequence of the largest repeat in EBV (IR1). We first characterized the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and we suggest that tumor-associated viruses may be more likely to contain DNA mixed from two strains. The patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.

Abstract 4822: Detection of EBV BamHI W region in surgical cancer specimen is a useful method to evaluate the risk of lymphomagenesis in patient-derived tumor xenografts

Abstract Patient-derived tumor xenografts (PDXs) established by xenotransplantation of surgically-resected human cancer specimens are an attractive model to analyze the character of cancer cells within the patient cancer tissues. However, establishment of PDX is occasionally hampered by lymphomagenesis. Lymphomagenesis in the PDX has been observed in the xenotransplantation of several types of cancers, such as lung, liver, gastric, bladder, breast, prostate, and colorectal cancers. Lymphomagenesis in the PDX is caused by the proliferation of the EBV-infected lymphocytes under insufficient immunosurveillance in the immunodeficient mice. However, it is still difficult to predict lymphomagenesis before xenotransplantation. In this study, we analyzed the expression of EBV-related genes in the 15 surgical specimens of the consented colorectal cancer (CRC) patients and the PDXs established by their xenotransplantation. The CRC specimens and the established PDX tumors were histologically examined. Then, the specimens were immunohistochemically stained with anti-CD3 and CD20 antibodies. The presence of EBV was histologically evaluated by EBER in situ. The expression levels of EBV-related genes, such as EBNAs, LMP1, EBER, BamHI W region and EBV-associated microRNAs, were evaluated using the genomic DNA and mRNAs prepared from the CRC specimens and the established PDX tumors. Nine PDXs were established by the xenotransplantation. Histological examination showed that 7 of 9 (78%) PDX recapitulated histopathological characteristics of the patient CRC tissues. However, 2 of 9 (22%) PDXs exhibited the morphological characteristics of EBV-associated human diffuse large B cell lymphoma (DLBCL). Then, we confirmed that lymphoma was formed by clonal proliferation of human B-cell lymphocytes, and strongly positive for EBER. We investigated whether the expression of EBV-related genes in the patient specimen is associated with lymphomagenesis in the PDXs. Expression of EBV genes and RNAs in patient CRC tissues were not clearly associated with lymphomagenesis in the PDXs. BamHI W region is a major internal repeat in EBV genome and the PCR amplification of this region is useful to evaluate the presence and amount of EBV. When the EBV BamHI W region was detectable in the patient specimens, the transplantation of the samples resulted in lymphomagenesis in 2 out of 3 patient cancer specimens. In contrast, when this region was undetectable, no patient cancer specimens resulted in lymphomagenesis (0 out of 7 patient cancer specimens). These results suggest that the amount and/or presence of EBV itself in the patient cancer specimens is one of the factors that are associated with lymphomagenesis in the PDXs. Therefore, the PCR amplification of EBV BamHI W region is an attractive method to evaluate the risk of lymphomagenesis before xenotransplantation. Citation Format: Junko Mukohyama, Dai Iwakiri, Yoh Zen, Toru Mukohara, Hironobu Minami, Yoshihiro Kakeji, Yohei Shimono. Detection of EBV BamHI W region in surgical cancer specimen is a useful method to evaluate the risk of lymphomagenesis in patient-derived tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4822. doi:10.1158/1538-7445.AM2017-4822

Evaluation of the risk of lymphomagenesis in xenografts by the PCR-based detection of EBV BamHI W region in patient cancer specimens.

Establishment of patient-derived tumor xenografts (PDXs) is hampered by lymphomagenesis mostly caused by the latently-infected Epstein-Barr virus (EBV) contained in patient cancer tissues. However, the character of patient tissues that result in lymphomagenesis after xenotransplantation is not elucidated. In this study, we analyzed the patient colorectal cancer (CRC) tissues and the PDXs established by their xenotransplantation. We found that 2 of 9 (22%) PDX tumors were EBV-associated human diffuse large B cell lymphoma which was formed by clonal proliferation of human B-cell lymphocytes, were strongly positive for EBER-ISH, and were classified as type III latency. Expression of EBV genes and RNAs, such as EBNAs, LMP1, EBER and EBV-associated microRNAs in patient CRC tissues were unlikely to be associated with lymphomagenesis in PDXs. In contrast, the positive PCR-based amplification of BamHI W region, a major internal repeat in EBV genome, in the patient CRC tissues was correlated with lymphomagenesis in PDXs. These results suggest that the detection of the EBV BamHI W region in the patient surgical specimens will be an effective way to predict the risk of lymphomagenesis in PDXs before xenotransplantation.

Variable Methylation of the Epstein-Barr Virus Wp EBNA Gene Promoter in B-Lymphoblastoid Cell Lines

During the initial stages of Epstein-Barr virus (EBV) infection of peripheral resting B cells, transcription of the six genes encoding the EBV latency-associated nuclear antigens (EBNAs) is driven from Wp, a promoter that is present in multiple copies within the EBV major internal repeat. As infection progresses, transcription from Wp is downregulated following upregulation of EBNA gene transcription driven from a promoter, Cp, located ca. 3 kb upstream of the first copy of Wp. Recently published data have provided evidence that, concomitant with the switch in EBNA gene promoter usage, Wp becomes heavily methylated (R. J. Tierney et al., J. Virol. 74:10468-10479, 2000). Based on this observation, it has been argued that methylation of Wp plays a pivotal role in suppressing Wp activity in EBV-immortalized B-lymphoblastoid cell lines (LCLs). Here we present data compiled from analyses of Wp methylation in eight randomly selected low-passage-number B-LCLs. These data demonstrate that there is considerable variability in Wp methylation, both between different cell lines and within clonal LCLs. Overall, less methylation of Wp was noted in established, low-passage-number LCLs than was previously observed in bulk cultures of infected B cells at days 18 and 21 postinfection. Importantly, the majority of LCLs examined harbored both unmethylated and methylated copies of Wp. In addition, all low-passage-number LCLs examined contained both Cp- and Wp-initiated EBNA transcripts, arguing for the presence of some transcriptionally active copies of Wp. Taken together, these data argue that other factors, perhaps in conjunction with Wp methylation, play a role in suppressing Wp activity in LCLs.

Deletion of Epstein-Barr virus regulatory sequences upstream of the EBNA gene promoter Wp1 is unfavorable for B-Cell immortalization.

Transcription of the six Epstein-Barr virus (EBV) EBNA genes is coordinately regulated, being driven by either the Cp promoter, which is encoded within the unique region just upstream of the EBV major internal repeat (IR-1), or by the Wp promoter, which is encoded within the IR-1 repeat and thus present in multiple copies. Previous analyses of Cp- and Wp-initiated transcription have identified a shared cis-regulatory element mapping to the region extending from -169 to -369 bp upstream of the Wp transcription initiation site (M. T. Puglielli, N. Desai, and S. H. Speck, J. Virol. 71:120-128, 1997). To assess the impact of this regulatory region on Cp and Wp activity in the context of the viral genome, we attempted to delete this regulatory region upstream of the first copy of Wp (Wp1). While 10 recombinant viruses were obtained in which this deletion was incorporated in the interior of the IR-1 repeat, only a single lymphoblastoid cell line (LCL) immortalized by a recombinant EBV harboring the deletion upstream of Wp1 was recovered. In contrast, using a control targeting vector in which the Wp regulatory sequences were intact but which contained a sequence tag within the W0 exon, we demonstrated that of the five recombinant viruses analyzed in which the crossover event had occurred upstream of the Wp sequence tag, four had incorporated the tagged sequences into Wp1 of the virus. Taken together, these results indicate that deletion of the regulatory sequences from -369 to -169 bp upstream of Wp1 is unfavorable for EBV-driven B-cell immortalization but is tolerated within the interior of the IR-1 repeat. Analysis of promoter usage in the clone 9-60 LCL, in which the W enhancer sequences were deleted upstream of Wp1, revealed the following: (i) the level of Cp-initiated transcription was significantly diminished compared to that of wild-type LCLs; (ii) the decreased Cp-initiated transcription was not efficiently compensated by transcription initiation from Wp1; and (iii) transcription initiation from downstream Wp promoters was detectable. This is the first report of an LCL in which transcription initiation from a Wp downstream of Wp1 has been documented.

Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses.

The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBNA2 is a key transcriptional regulator of both viral and cellular gene expression and is essential for EBV-induced immortalization of B lymphocytes. EBNA-LP is also important for EBV-induced immortalization of B lymphocytes, but far less is known about the functional domains and cellular cofactors that mediate EBNA-LP function. While recent studies suggest that serine phosphorylation of EBNA-LP and coactivation of EBNA2-mediated transactivation are important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. Therefore, we have used a comparative approach by studying the EBNA-LP homologues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid sequences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserved regions can be defined, and four of eight potential serine residues are conserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of upstream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rhesus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. The rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promoters through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5- to 12-fold. Thus, there is strong structural and functional conservation among the simian EBNA-LP homologues. Identification of evolutionarily conserved serine residues and regions in EBNA-LP homologues provides important clues for identifying the cellular cofactors and molecular mechanisms mediating these conserved viral functions.

B-cell lines immortalized with an Epstein-Barr virus mutant lacking the Cp EBNA2 enhancer are biased toward utilization of the oriP-proximal EBNA gene promoter Wp1

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities have previously been shown to be mutually exclusive in established lymphoblastoid cell lines. Initially after infection, the EBNA genes are transcribed from Wp, which is present in multiples copies within the major internal repeat of EBV. Approximately 48 to 72 h postinfection, Wp is downregulated, with a corresponding increase in transcription from Cp. An EBNA2-responsive enhancer exists upstream of Cp, and a role for EBNA2 in the induction of Cp activity during the establishment of viral latency has previously been proposed (Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 87:1725-1729, 1991). To critically assess the potential role for this enhancer region in determining relative usage of Cp and Wp, an EBNA2 enhancer deletion mutant virus was generated. Lymphoblastoid cell lines were screened by PCR and Southern blotting for the presence of mutant virus harboring the EBNA2 enhancer deletion. A quantitative S1 nuclease protection assay was developed to allow comparison of relative Cp and Wp activities for the cell lines containing mutant virus and those of the wild-type recombinants which lacked the enhancer deletion. In general, the wild-type recombinants had higher levels of Cp-initiated transcripts than Wp-initiated transcripts. In contrast, the Cp EBNA2 enhancer deletion mutants exhibited a strong bias toward Wp activity. Notably, only the first Wp (oriP-proximal Wp; Wp1) appears active in these mutants. S1 nuclease protection assays using a probe which hybridizes to the W2 exon, contained in both Cp- and Wp-initiated transcripts, indicated that the total level of transcription from Cp and Wp remained the same in wild-type and EBNA2 enhancer mutant cell lines. The presence of both Cp and Wp activity in the wild-type recombinants, as well as in newly derived lymphoblastoid cell lines established with the prototype B95.8 virus, demonstrated that Cp and Wp activities are not always mutually exclusive.

Alternative splicing dictates translational start in Epstein-Barr virus transcripts.

Of the 10 viral genes known to be expressed during Epstein-Barr virus (EBV) latency, six encode nuclear antigens (EBNAs), all of which are expressed from a long primary transcript by means of alternative splicing and alternative polyadenylation sites. The primary transcript is generated by either of two promoters which operate in a mutually exclusive fashion in different clonal cell lines. All mRNAs from either promoter have exons in common from the BamHI W viral genomic fragment (the major internal repeat, IR1) which encode the N-terminal portion of one of the nuclear antigens (EBNA 4). In addition to the coding regions for EBNA 4, EBNA mRNAs encode another EBNA (i.e. EBNA 1, 2, 3A, 3B or 3C) downstream. We show that alternative splicing determines whether the translation initiation codon for EBNA 4 is present or absent, thus permitting the generation of mRNAs in which the first translation initiation codon is either that for the EBNA 4 gene or for the other EBNA gene encoded downstream. This mechanism presumably ensures efficient translation of all the EBNA genes.

Bidirectional transcription of the Epstein-Barr virus major internal repeat

In latently infected cell lines, the Epstein-Barr virus BamHI W fragment (major internal repeat) is transcribed in a rightward direction to yield exons common to several alternatively spliced messages which encode the six known viral nuclear antigens. A substantial steady-state population of very large (up to 20-kilobase) rightward transcripts is nuclear, much of it being polyadenylated. We report a rise in the levels of rightward transcripts hybridizing to BamHI-W sequences upon phorbol ester treatment of the clone-13 Burkitt's lymphoma cell line. We also report large (up to 15-kilobase) leftward transcripts hybridizing to BamHI-W sequences which occurred late in the viral lytic cycle in B95-8 and clone-13 cells. These leftward transcripts may antagonize the expression of the viral nuclear antigen messages by the formation of RNA duplexes.

Size and stability of the Epstein-Barr virus major internal repeat (IR-1) in Burkitt's lymphoma and lymphoblastoid cell lines

We have used field inversion gel electrophoresis to survey EBV strains for the size of the major internal repeat, IR-1, and estimate the number of 3.1-kb repeat units present. The B95-8 strain of EBV was estimated to contain 8.6 repeats. The repeat number varies considerably among naturally occurring isolates around a mean of six repeats. Some cell lines harbored multiple viral genomes with differing numbers of repeats and our results suggest that the repeat number in IR-1 is more likely to change during lytic replication than during latency. The Jijoye strain had 6.6 repeats and the Jijoye deletion mutant clone P3HR-1 retained 5.9 repeats setting the size of the P3HR-1 deletion at 6.8 kb. Thus, the nonimmortalizing mutant has retained all of the W1 and W2 exons of the immortalizing parent and has lost only the 3′ unique exons of EBNA4 and all of EBNA2.

Complex transcription of the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) in latently and lytically infected B lymphocytes.

Several cDNA clones containing the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) have been recovered from the tightly latent lymphoblastoid cell line IB4. These clones contain the 5' leader exons encoded in the major internal repeat 1 and the viral BamHI fragment Y, identified in the rightwardly transcribed viral mRNAs associated with the latent viral life cycle. In addition, a cDNA clone containing BHRF1 from the Burkitt lymphoma cell line Jijoye was also recovered and exhibits a distinctive splicing pattern. In vitro transcription and translation of BHRF1, followed by immunoprecipitation with Epstein-Barr virus-positive human sera, indicates that this viral antigen is expressed during infection. RNA blot analyses with a wide panel of lymphoblastoid and Burkitt lymphoma cell lines revealed a complex pattern of transcription. Hybridization data obtained with several probes is presented.

An Epstein-Barr virus transcript from a latently infected, growth-transformed B-cell line encodes a highly repetitive polypeptide.

By screening a cDNA library prepared from poly(A)+ RNA isolated from an Epstein-Barr virus latently infected, growth-transformed human B-lymphoblastoid cell line, we have recovered a clone corresponding to a highly spliced viral transcript encoded largely by the major internal repeat (IR1). The 5' region contains one copy of a 26-base-pair (bp) exon (W0) [which is 28 bp downstream from a CAATT-(N)34TATAAA sequence (N, unspecified base)] and seven copies of two small exons (W1, 66 bp; W2, 132 bp). In addition, there are three exons from the "unique" region of the BamHI Y fragment of the viral genome. Two other cDNA clones that have been described, corresponding to latent viral transcripts, share homology in their 5' regions with this clone and are clearly divergent at their 3' ends. The cDNA clone described in this paper contains one long open reading frame that extends through the repeat element. In vitro transcription and translation of this open reading frame yielded a 62-kDa polypeptide that could be immunoprecipitated by an Epstein-Barr virus-positive human serum.