Characterising a human endogenous retrovirus(HERV)-derived tumour-associated antigen: enriched RNA-Seq analysis of HERV-K(HML-2) in mantle cell lymphoma cell lines.
ABSTRACT: Background:The cell-surface attachment protein (Env) of the HERV-K(HML-2) lineage of endogenous retroviruses is a potentially attractive tumour-associated antigen for anti-cancer immunotherapy. The human genome contains around 100 integrated copies (called proviruses or loci) of the HERV-K(HML-2) virus and we argue that it is important for therapy development to know which and how many of these contribute to protein expression, and how this varies across tissues. We measured relative provirus expression in HERV-K(HML-2), using enriched RNA-Seq analysis with both short- and long-read sequencing, in three Mantle Cell Lymphoma cell lines (JVM2, Granta519 and REC1). We also confirmed expression of the Env protein in two of our cell lines using Western blotting, and analysed provirus expression data from all other relevant published studies. Results:Firstly, in both our and other reanalysed studies, approximately 10% of the transcripts mapping to HERV-K(HML-2) came from Env-encoding proviruses. Secondly, in one cell line the majority of the protein expression appears to come from one provirus (12q14.1). Thirdly, we find a strong tissue-specific pattern of provirus expression. Conclusions:A possible dependency of Env expression on a single provirus, combined with the earlier observation that this provirus is not present in all individuals and a general pattern of tissue-specific expression among proviruses, has serious implications for future HERV-K(HML-2)-targeted immunotherapy. Further research into HERV-K(HML-2) as a possible tumour-associated antigen in blood cancers requires a more targeted, proteome-based, screening protocol that will consider these polymorphisms within HERV-K(HML-2). We include a plan (and necessary alignments) for such work.
Project description:BACKGROUND: A significant proportion of the human genome is comprised of human endogenous retroviruses (HERVs). HERV transcripts are found in every human tissue. Expression of proviruses of the HERV-K(HML-2) family has been associated with development of human tumors, in particular germ cell tumors (GCT). Very little is known about transcriptional activity of individual HML-2 loci in human tissues, though. RESULTS: By employing private nucleotide differences between loci, we assigned approximately 1500 HML-2 cDNAs to individual HML-2 loci, identifying, in total, 23 transcriptionally active HML-2 proviruses. Several loci are active in various human tissue types. Transcription levels of some HML-2 loci appear higher than those of other loci. Several HML-2 Rec-encoding loci are expressed in GCT and non-GCT tissues. A provirus on chromosome 22q11.21 appears strongly upregulated in pathologic GCT tissues and may explain high HML-2 Gag protein levels in GCTs. Presence of Gag and Env antibodies in GCT patients is not correlated with activation of individual loci. HML-2 proviruses previously reported capable of forming an infectious HML-2 variant are transcriptionally active in germ cell tissue. Our study furthermore shows that Expressed Sequence Tag (EST) data are insufficient to describe transcriptional activity of HML-2 and other HERV loci in tissues of interest. CONCLUSION: Our, to date, largest-scale study reveals in greater detail expression patterns of individual HML-2 loci in human tissues of clinical interest. Moreover, large-scale, specialized studies are indicated to better comprehend transcriptional activity and regulation of HERVs. We thus emphasize the need for a specialized HERV Transcriptome Project.
Project description:To date, the human population census of proviruses of the Betaretrovirus-like human endogenous retroviral (HERV-K) (HML-2) family has been compiled from a limited number of complete genomes, making it certain that rare polymorphic loci are under-represented and are yet to be described.Here we describe a suppression PCR-based method called genome-wide amplification of proviral sequences (GAPS) that selectively amplifies DNA fragments containing the termini of HERV-K(HML-2) proviral sequences and their flanking genomic sequences. We analysed the HERV-K(HML-2) proviral content of 101 unrelated humans, 4 common chimpanzees and three centre d'etude du polymorphisme humain (CEPH) pedigrees (44 individuals). The technique isolated HERV-K(HML-2) proviruses that had integrated in the genomes of the great apes throughout their divergence and included evolutionarily young elements still unfixed for presence/absence.By examining the HERV-K(HML-2) proviral content of 145 humans we detected a new insertionally polymorphic Type I HERV-K(HML-2) provirus. We also observed provirus versus solo long terminal repeat (LTR) polymorphism within humans at a previously unreported, but ancient, locus. Finally, we report two novel chimpanzee specific proviruses, one of which is dimorphic for a provirus versus solo LTR. Thus GAPS enables the isolation of uncharacterised HERV-K(HML-2) proviral sequences and provides a direct means to assess inter-individual genetic variation associated with HERV-K(HML-2) proviruses.
Project description:Increasing evidence suggests that repetitive elements may play a role in host gene regulation, particularly through the donation of alternative promoters, enhancers, splice sites, and termination signals. Elevated transcript expression of the endogenous retrovirus group HERV-K (HML-2) is seen in many human cancers, although the identities of the individual proviral loci contributing to this expression as well as their mechanisms of activation have been unclear. Using high-throughput next-generation sequencing techniques optimized for the capture of HML-2 expression, we characterized the HML-2 transcriptome and means of activation in an in vitro model of human mammary epithelial cell transformation. Our analysis showed significant expression originating from 15 HML-2 full-length proviruses, through four modes of transcription. The majority of expression was in the antisense orientation and from proviruses integrated within introns. We found two instances of long terminal repeat (LTR)-driven provirus transcription but no evidence to suggest that these active 5' LTRs were influencing nearby host gene expression. Importantly, LTR-driven transcription was restricted to tumorigenic cells, suggesting that LTR promoter activity is dependent upon the transcriptional environment of a malignant cell.IMPORTANCE Here, we use an in vitro model of human mammary epithelial cell transformation to assess how malignancy-associated shifts in the transcriptional milieu of a cell may impact HML-2 activity. We found 15 proviruses to be significantly expressed through four different mechanisms, with the majority of transcripts being antisense copies of proviruses located within introns. We saw active 5' LTR use in tumorigenic cells only, suggesting that the cellular environment of a cancer cell is a critical component for induction of LTR promoter activity. These findings have implications for future studies investigating HML-2 as a target for immunotherapy or as a biomarker for disease.
Project description:The human germ cell tumor line Tera-1 produces retroviral particles which are encoded by the human endogenous retrovirus family HERV-K(HML-2). We show here, by quantitative reverse transcriptase PCR, that HML-2 gag and env RNA transcripts are selectively packaged into Tera-1 retroviral particles, whereas RNAs from cellular housekeeping genes and from other HERV families (HERV-H and HERV-W) are nonselectively copackaged. Assignment of cloned HML-2 gag and env cDNAs from Tera-1 retroviral particles to individual HML-2 loci in the human genome demonstrated that HML-2 RNA transcripts packaged into Tera-1 retroviral particles originate almost exclusively from an HML-2 provirus on chromosome 22q11.21. Based on relative cloning frequencies, this provirus was the most active among a total of eight transcribed HML-2 loci identified in Tera-1 cells. These data suggest that at least one HML-2 element, that is, the HML-2 provirus on 22q11.21, has retained the capacity for packaging RNA into HML-2-encoded retroviral particles. Given its elevated transcriptional activity and the presence of a full-length Gag open reading frame, the 22q11.21 HML-2 provirus may also significantly contribute to Gag protein and thus particle production in Tera-1 cells. Our findings provide important clues to the generation and biological properties of HML-2-encoded particles. In addition, copackaging of non-HML-2 HERV transcripts in HML-2-encoded particles should inform the debate about endogenous retroviral particles putatively encoded by non-HML-2 HERV families that have previously been described for other human diseases, such as multiple sclerosis.
Project description:Recent studies suggest that human endogenous retrovirus group K (HERV-K) provirus expression plays a role in the pathogenesis of HIV-1 infection. In particular, RNA from the HML-2 subgroup of HERV-K proviruses has been reported to be highly expressed at the cellular level and detectable in the plasma of HIV-1-infected patients, suggestive of virion production and, perhaps, replication. In this study, we developed an HML-2-specific quantitative-PCR assay that detects 51 of the 89 known HML-2 proviruses in the human genome. Plasma and peripheral blood mononuclear cells (PBMCs) from HIV-negative controls and HIV-1-infected patients were collected for analysis of HML-2 RNA expression. Contrary to previous reports, we did not detect high levels of HML-2 RNA in the plasma of HIV-1-infected patients, but we did observe a significant increase of HML-2 RNA in total PBMCs compared to HIV-negative controls. The level of HML-2 expression in PBMCs does not appear to be related to patient use of antiretrovirals or to HIV-1 plasma RNA, cellular RNA, or cellular DNA levels. To investigate the source of HML-2 RNA expression, patient PBMCs were sorted into CD3+ CD4+, CD3+ CD8+, CD3- CD14+, and CD3- CD20+ cell subsets and then analyzed for HML-2 RNA levels. No single cell subset was enriched for HML-2 RNA expression in HIV-1-infected patients, but there appears to be substantial variability in the level of HML-2 expression depending on the cell type.Here, we report that human endogenous retrovirus group K (HERV-K) (HML-2) proviruses are expressed at significantly higher levels in peripheral blood mononuclear cells (PBMCs) from patients with HIV-1 infection than in those from uninfected individuals. However, contrary to previous reports, this expression did not lead to detectable virions in the plasma of these patients. In addition, we found that HML-2 proviruses were expressed in multiple blood cell types from HIV-1-infected individuals, and the magnitude of HML-2 expression was not related to HIV-1 disease markers in this patient cohort. These findings may have implications for HML-2-based therapies targeting HIV-1 infection.
Project description:Human endogenous retrovirus-K (HERV-K) human mouse mammary tumor virus-like 2 (HML-2) is the most recently active endogenous retrovirus group in humans, and the only group with human-specific proviruses. HML-2 expression is associated with cancer and other diseases, but extensive searches have failed to reveal any replication-competent proviruses in humans. However, HML-2 proviruses are found throughout the catarrhine primates, and it is possible that they continue to infect some species today. To investigate this possibility, we searched for gorilla-specific HML-2 elements using both in silico data mining and targeted deep-sequencing approaches. We identified 150 gorilla-specific integrations, including 31 2-LTR proviruses. Many of these proviruses have identical LTRs, and are insertionally polymorphic, consistent with very recent integration. One identified provirus has full-length ORFs for all genes, and thus could potentially be replication-competent. We suggest that gorillas may still harbor infectious HML-2 virus and could serve as a model for understanding retrovirus evolution and pathogenesis in humans.
Project description:A significant proportion of the human genome consists of stably inherited retroviral sequences. Most human endogenous retroviruses (HERVs) became defective over time. The HERV-K(HML-2) family is exceptional because of its coding capacity and the possible involvement in germ cell tumor (GCT) development. HERV-K(HML-2) transcription is strongly upregulated in GCTs. However, regulation of HERV-K(HML-2) transcription remains poorly understood. We investigated in detail the role of CpG methylation on the transcriptional activity of HERV-K(HML-2) long terminal repeats (LTRs). We find that CpG sites in various HERV-K(HML-2) proviral 5' LTRs are methylated at different levels in the cell line Tera-1. Methylation levels correlate with previously observed transcriptional activities of these proviruses. CpG-mediated silencing of HERV-K(HML-2) LTRs is further corroborated by transcriptional inactivity of in vitro-methylated 5' LTR reporter plasmids. However, CpG methylation levels do not solely regulate HERV-K(HML-2) 5' LTR activity, as evidenced by different LTR activities in the cell line T47D. A significant number of mutated CpG sites in evolutionary old HERV-K(HML-2) 5' LTRs suggests that CpG methylation had already silenced HERV-K(HML-2) proviruses millions of years ago. Direct silencing of HERV-K(HML-2) expression by CpG methylation enlightens upregulated HERV-K(HML-2) expression in usually hypomethylated GCT tissue.
Project description:The human genome harbors numerous distinct families of so-called human endogenous retroviruses (HERV) which are remnants of exogenous retroviruses that entered the germ line millions of years ago. We describe here the hitherto little-characterized betaretrovirus HERV-K(HML-5) family (named HERVK22 in Repbase) in greater detail. Out of 139 proviruses, only a few loci represent full-length proviruses, and many lack gag protease and/or env gene regions. We generated a consensus sequence from multiple alignment of 62 HML-5 loci that displays open reading frames for the four major retroviral proteins. Four HML-5 long terminal repeat (LTR) subfamilies were identified that are associated with monophyletic proviral bodies, implying different evolution of HML-5 LTRs and genes. Sequence analysis indicated that the proviruses formed approximately 55 million years ago. Accordingly, HML-5 proviral sequences were detected in Old World and New World primates but not in prosimians. No recent activity is associated with this HERV family. We also conclude that the HML-5 consensus sequence primer binding site is identical to methionine tRNA. Therefore, the family should be designated HERV-M. Our study provides important insights into the structure and evolution of the oldest betaretrovirus in the primate genome known to date.
Project description:BACKGROUND: Malignant human embryonal carcinoma cells (ECCs) rely on similar transcriptional networks as non-malignant embryonic stem cells (ESCs) to control selfrenewal, maintain pluripotency, and inhibit differentiation. Because re-activation of silenced HERV-K(HML-2) loci is a hallmark of ECCs, we asked if this HERV group was also reactivated in ESCs and induced pluripotent stem cells (iPSCs). FINDINGS: Using RT-PCR and Western Blot, we demonstrate HERV-K(HML-2) RNA and protein expression in undifferentiated human ESCs and iPSCs. Induction of differentiation by embryoid body formation resulted in rapid silencing of HERV-K(HML-2) provirus expression. Sequencing analysis of a conserved region of the gag gene showed that proviral expression in ESCs and iPSCs represents at least 11 of the 66 nearly full length HERV-K(HML-2) loci, with slightly varying patterns in individual cell lines. These proviruses are human specific integrations and harbor promoter competent long terminal repeats (LTR5hs subgroup). We observed high mRNA levels of the NP9 and Gag encoding proviruses K101(22q11.21) in all and K10(5q33.3) in most of the ECC, ESC, and iPSC lines tested, while K37(11q23.3) mRNA was detected only in ESCs and iPSCs. In addition, we detected expression of proviral mRNA encoding the RNA export adaptor Rec in all cell lines studied. Proviral mRNA originating from the K108(7p22.1) locus, which inter alia codes for functional Rec and Env proteins, was only reactivated in malignant ECC lines, not in benign ESCs or iPSCs. CONCLUSIONS: HERV-K(HML-2) RNA and protein expression is a marker for pluripotent human stem cells. Initiation of differentiation results in rapid down-regulation. Further studies are needed to explore a putative functional role of HERV-K(HML-2) RNA and proteins in pluripotent stem cells.
Project description:BACKGROUND: The human genome comprises numerous human endogenous retroviruses (HERVs) that formed millions of years ago in ancestral species. A number of loci of the HERV-K(HML-2) family are evolutionarily much younger. A recent study suggested an infectious HERV-K(HML-2) variant in humans and other primates. Isolating such a variant from human individuals would be a significant finding for human biology. RESULTS: When investigating expression patterns of specific HML-2 proviruses we encountered HERV-K(HML-2) cDNA sequences without proviral homologues in the human genome, named HERV-KX, that could very well support recently suggested infectious HML-2 variants. However, detailed sequence analysis, using the software RECCO, suggested that HERV-KX sequences were produced by recombination, possibly arising ex vivo, between transcripts from different HML-2 proviral loci. CONCLUSION: As RT-PCR probably will be instrumental for isolating an infectious HERV-K(HML-2) variant, generation of "new" HERV-K(HML-2) sequences by ex vivo recombination seems inevitable. Further complicated by an unknown amount of allelic sequence variation in HERV-K(HML-2) proviruses, newly identified HERV-K(HML-2) variants should be interpreted very cautiously.