Human endogenous retroviruses (HERVs) are remnants of ancient retroviruses, which have infected and been integrated into the host genome (Weiss, 2006; Katzourakis et al., 2009). HERVs belong to transposable elements (TEs) based on the “copy and paste” life cycle of replicative transposition (Weiss, 2006; Katzourakis et al., 2009). A substantial number of HERVs have been retained in human genome during the long evolutionary history and constitute approximately 8% of human genome (Weiss, 2006). HERVs were previously considered “junk DNA” sequences (Weiss, 2006). Increasing findings suggest that HERVs play a crucial role in immune response, placental morphology, cancer development, and aging processes (Mi et al., 2000; Deniz et al., 2020; Wilson et al., 2020; Liu et al., 2023; Ng et al., 2023). For example, a recent study has demonstrated the presence of HERV-K (HML-2) envelope-reactive antibodies in a large proportion of patients with lung adenocarcinoma (LUAD) and the antibodies could promote lung cancer immunotherapy (Ng et al., 2023). It has been documented that syncytin is the envelope protein derived from HERVs and essential for placental morphogenesis (Mi et al., 2000). And emerging studies have further showed that HERVs participate in the expression regulation of placenta-associated genes (Frost et al., 2023). The effect of HERVs on cellular aging has also been discovered and the reactivation of the youthful HERV-K triggers cellular senescence and tissue aging (Liu et al., 2023). And HERVs have been proposed to serve as a potential target to alleviate aging (Liu et al., 2023). Apart from the function mentioned above, numbers of findings have suggested that HERVs are heavily implicated in occurrence and development of various cancers (Kitsou et al., 2023). One of classic examples is that the accessary protein Np9 is a critical molecular switch of multiple signaling pathways and could promote the growth of human leukemia stem/progenitor cells (Chen et al., 2013).

The complete proviral genome structure of HERVs consists of gag, pro, pol, and env genes flanked by long terminal repeats (LTRs), which usually serve as regulatory elements encompassing the TATA box and transcription factor binding sites (TFBSs) (Hurst and Magiorkinis, 2017). Due to the accumulation of numerous mutations, insertions and deletions (INDELs), most HERVs gradually lose the coding ability during evolution. Moreover, internal coding regions are deleted and the remaining LTR sequences eventually form solitary LTRs (solo-LTRs) through homologous recombination (Cohen et al., 2009). It has been reported that solo-LTRs are the most common HERV traces (Vargiu et al., 2016), accounting for about 90% of all HERV insertions (Subramanian et al., 2011; Thomas et al., 2018). The complete LTR elements comprise three conserved regions, namely U3, R and U5 (Vargiu et al., 2016). The U3 regions usually encompass promoter and enhancer elements that modulate the expression of internal genes (Vargiu et al., 2016). In most cases, the transcription initiation site (TSS) could be identified in the 5′ terminal R region while the 3′ terminal R region contains a polyadenylation (polyA) signaling site (Gifford and Tristem, 2003). Although solo-LTRs lose the internal genes, numerous studies have demonstrated the impact of solo-LTRs on the expression of adjacent genes (Ruda et al., 2004; Romanish et al., 2007; Conley et al., 2008; Cohen et al., 2009; Babaian and Mager, 2016). And solo-LTRs are also able to regulate the expression of distal genes as enhancers (Pi et al., 2004; Pi et al., 2010; Deniz et al., 2020). The exaptation of human endogenous retroviral promoters and enhancers has driven tissue-specific and lineage-specific patterns of gene expression and plays a substantial effect on diverse human biology processes, especially carcinogenesis and development (Conley et al., 2008; Cohen et al., 2009; Babaian and Mager, 2016; Hurst and Magiorkinis, 2017) (Figure 1A).

Various studies have documented the function of long non-coding RNAs (lncRNAs) in multiple biological processes including embryonic development, cell proliferation and immune response at the transcriptional and post-transcriptional levels (Statello et al., 2021). Emerging studies have revealed that active solo-LTRs could be transcribed and give rise to lncRNAs (Figure 1B), which are closely linked with multiple stages of tumorigenesis and metastasis (Prensner et al., 2013; Jin et al., 2019; Wu et al., 2020). These lncRNAs have been suggested as potential biomarkers for clinical cancer diagnosis and treatment (Prensner et al., 2013; Jin et al., 2019; Wu et al., 2020). For example, TROJAN, one solitary LTR70-derived lncRNA, promotes triple-negative breast cancer (TNBC) progression through ZMYND8 degradation (Jin et al., 2019).

In this review, we provide a window of the roles of solo-LTRs in human health and disease. Here we will illustrate the regulatory function of solo-LTRs demonstrated in a number of studies. Additionally, we will focus on the emerging discoveries of solo-LTRs-derived genes (mainly mRNAs and lncRNAs), exploring their expression patterns and regulatory mechanisms in diverse diseases. Importantly, these findings underscore the significance of the abundant reservation of solo-LTRs within human genome, particularly highlighting the potential involvement in cancer occurrence and progression.

Based on phylogenetic evidence of pol gene, endogenous retroviruses (ERVs) have been classified into three main classes (Gifford and Tristem, 2003). Class I ERVs include gamma- and epsilonretroviruses, class II comprises alpha-, beta-, deltaretroviruses, and lentiviruses, and class III ERVs include spumaviruses (Gifford and Tristem, 2003). A specific tRNA hybridizes with the primer binding sites (PBS) of HERVs to initiate the reverse transcription reaction (Bannert and Kurth, 2006; Johnson, 2015). According to the identity of the 18 nucleotides in the PBS, HERVs are also classified into many families (Bannert and Kurth, 2006). For example, HERV-H family contains the PBS with a sequence similar to a histidine (H) tRNA, and those assumed to use tRNAHis would be also assigned to the HERV-H family (Bannert and Kurth, 2006). Thus, three ERV classes are grouped into diverse families (Bannert and Kurth, 2006; Johnson, 2015). Class I HERVs include HERV-H, HERV-W, HERV-E, HERV-F, HERV-I, HERV-P, HERV-T and so on. Class II HERVs have only one HERV-K family. And HERV-R and HERV-S are members of class III HERVs.

Due to the advent of next-generation sequencing technologies, several bioinformatics tools have been designed to detect polymorphic ERV insertions in human genome such as RetroTector (Sperber et al., 2007), RetroSeq (Keane et al., 2013) and ERVcaller (Chen and Li, 2019). Compared to existing tools such as RetroSeq, ERVcaller based on sequence alignment achieves both the higher sensitivity and precision for detecting simulated ERV and other TE insertions (Chen and Li, 2019). And ERVcaller can be applied broadly to other species other than human (Chen and Li, 2019). But the existing tools still have some limitations. For instance, ERVcaller requires ERV references (Chen and Li, 2019). Novel methods and softwares for identifying polymorphic ERVs are in need to be developed.

Databases describing the structure and distribution of HERVs have also been developed such as RepBase (Bao et al., 2015), Dfam (Storer et al., 2021) and HERVd (Paces et al., 2002). There are 770,551 HERV loci in human genome according to the annotation of RepeatMasker (http://repeatmasker.org) in UCSC table browser (https://genome.ucsc.edu/cgi-bin/hgTables) (Karolchik et al., 2004). The most common HERV traces are fragmented solo-LTRs while only 3,173 relatively intact HERV sequences were identified (Vargiu et al., 2016). It is an intriguing challenge to accurately classify the full spectrum of HERV insertions (particularly solo-LTRs) within related families.

As the high-throughput sequencing technology allows the obtaining of vertebrate genomes including a number of ERV sequences, a unified nomenclature system for ERV loci is urgently needed. A nomenclature system for retroviruses and ERVs proposed that the ERV loci should be composed of three elements, which include the lineage of retrovirus, a numeric ID and the host lineage (Gifford et al., 2018). For instance, the known full-length provirus HERV-K113, a member in HERV-K family (Jha et al., 2009), is designated as ERV-K(HML2).113-Hsa. This could contribute to the human genome annotation and the studies on pathophysiology function of HERVs.

Numbers of studies have showed that HERVs are an abundant reservoir of promoters and enhancers that can initiate or regulate transcription of adjacent genes in human health and diseases especially various cancers and the majority of HERV regulatory sequences belong to LTR elements, many of which are solitary LTRs (van de Lagemaat et al., 2003; Conley et al., 2008; Cohen et al., 2009; Babaian and Mager, 2016). In addition to triggering the expression of novel genes, LTRs could function as alternative promoters different from the native promoters (van de Lagemaat et al., 2003; Conley et al., 2008; Cohen et al., 2009; Babaian and Mager, 2016). Several studies have also documented the cell-specific or tissue-specific transcriptional regulation of LTR elements (van de Lagemaat et al., 2003; Conley et al., 2008; Cohen et al., 2009). For example, expression of β-galactoside α-2,6-sialyltransferase I (ST6Gal1), a glycan-modifying enzyme mediating the attachment of α-2,6-sialic acids, is modulated by three different promoters, generating three mRNA isoforms, which have been identified in the liver, mature B-cells and a number of cell types, respectively (Aas-Eng et al., 1995; Milflores-Flores et al., 2012). Intriguingly, the expression of the transcript in mature B-cells is induced by a promoter, which includes several solo-LTRs (one MER4D element, two MER21A elements, and part of one MER21A element) (Lo and Lau, 1996) (Figure 2A, Supplementary Table S1). Further investigation showed that the solo-LTR sequences contain a number of known transcription factor binding sites (two AP2, two C/EBP and two NF-IL6) and the TATA box (Lo and Lau, 1996). It has been confirmed that the expression of ST6GAL1 in B cells is classically implicated in the dysregulated B cell development and immunoglobulin levels of ST6GAL1-deficient mice (Irons et al., 2020). So these solo-LTRs may play an important role in immune regulation through modulating the transcription of ST6GAL1. Another known example of the LTR with tissue-specific promoter and enhancer activities is LTR L47334, a solitary HERV-K LTR (Ruda et al., 2004). The promoter activity of the LTR was relatively weak in renal cell carcinoma GS and acute T cell leukemia Jurkat cells while a high promoter activity was detected in Tera-1 cells (Ruda et al., 2004). The enhancer activity of the LTR was only detected in Tera-1 cells and not in closely related NT2/D1 cells (Ruda et al., 2004).

The enrichment of multiple TFBSs in LTR elements is critical for transcriptional regulatory networks and phenotypic heterogeneity. A previous study has revealed that a significant proportion (32.1%) of the tumor suppressor protein p53 binding sites are embedded in LTR elements of ERV1 superfamily (LTR10B1, LTR10E, MER61E, LTR10D, and MER61C) (Bourque et al., 2008). For LTR9B elements from ERV1 superfamily, 33.2% of these repeats are bound by the key regulatory protein OCT4 in human embryonic stem cells (Kunarso et al., 2010). One of the notable examples is the solitary LTR of ERV-9 family. The LTR is located upstream of the human β-globin locus (Yu et al., 2005) (Figure 2B, Supplementary Table S1). The U3 region of the LTR element contains 14 tandemly repeated subunits with recurrent CCAAT and GATA motifs (Yu et al., 2005) (Figure 2B). The CCAAT motif could bind the ubiquitous transcription factor NF-Y while the GATA motif binds the GATA family of transcription factors, including GATA-1 and -2 (Yu et al., 2005). NF-Y bound at the CCAAT motif recruits GATA-1 and -2, thus generating a novel LTR enhancer-pol II complex (Yu et al., 2005; Hu et al., 2017). Furthermore, synthesis of lncRNA from the 5′ end of the R region through the U5 region into the downstream genomic DNA is initiated by the LTR enhancer complex (Hu et al., 2017). The lncRNA could act in cis to modulate long-range LTR enhancer function to activate transcription of downstream genes critical to erythropoiesis (Hu et al., 2017). In addition, about 80% of the LTRs among the 50 most highly expressed HERV-H proviruses are bound by core transcription factors associated with pluripotency, including POU class 5 homeobox 1 (OCT3/4), sex determining region Y-box 2box 2 (SOX2), and NANOG homeobox (NANOG) (Ohnuki et al., 2014). It has been demonstrated that LTR7, the LTR element of HERV-H, could promote cell reprogramming toward induced pluripotent stem cells (iPSCs) through being bound and activated by OCT3/4, SOX2, and KLF4 (Ohnuki et al., 2014). Therefore, LTR elements play a key role in expanding the repertoire of TFBSs and allowing a fine regulation of cellular biological processes.

It has been inferred that HERVs likely exert a slight effect on gene expression (Cohen et al., 2009). But to our knowledge, HERVs, notably LTR elements, contribute greatly to the regulatory networks of human genes. Much research efforts are dedicated to describe the effect of LTR-derived regulatory elements on human diseases especially cancers, and multiple findings have been reported including inducing the expression of oncogenes (Lamprecht et al., 2010; Nguyen et al., 2019), triggering the transcription of HERVs-derived double-stranded RNAs (dsRNA) associated with immune response (Zhou et al., 2021), and modulating the expression level of adjacent transcription factors (Ito et al., 2020; Zhou et al., 2021). In addition, there are increasing evidences that LTRs could be derepressed and play a role in shaping placental development (Yu et al., 2023), favoring immune response and driving human evolution (Jung et al., 2017; Srinivasachar Badarinarayan et al., 2020).

In previous studies, the identification of expressed HERVs was largely restricted due to the nature of repeat sequences. The application of high-throughput sequencing approaches has allowed to solve these problems, and individual HERV loci expressed in different cell types and tissues have gradually been found and elucidated. Numerous studies have demonstrated solo-LTR-initiated chimeric gene transcripts (mainly mRNAs and lncRNAs), which contribute greatly to human health and diseases (Cohen et al., 2009; Prensner et al., 2013; Gibb et al., 2015; Wiesner et al., 2015). And the solo-LTR-derived genes have been suggested as potential diagnostic and prognostic biomarkers across diverse cancer types. Therefore, studying solo-LTR-derived genes holds significant importance for tumor biology.

Solo-LTRs make up a large majority of the HERVs persisting in the human genome. Previous studies have shown that some of the many thousands of solo-LTRs are involved in various biological processes by acting as promoters/enhancers. In this review, we summarized the recent discoveries about the functions of a few solo-LTRs and the chimeric genes (especially lncRNAs) stemming from them and proximal sequences, thereby gradually uncovering the mechanisms and important functions of these few solo-LTRs. And the different pathways also reflect the complexity of solo-LTR-derived genes (Figure 5), which could affect tumor biology greatly. This review aims to highlight that solo-LTRs can alter transcription regulation of adjacent genes by providing enhancers and promoters and also shape the human transcriptome through generating lncRNAs. Although the functions of small quantity of solo-LTRs have been revealed and the precise mechanism and effect of a bulk of solo-LTRs activation remains to be uncovered, there is no doubt that solo-LTRs play a pivotal role in human health and disease. Further exploration to unveil the pathological processes and underlying mechanisms of solo-LTRs is urgently needed.