Regulation of PERVs
Although in most normal healthy cells of pigs, the release of PERV particles (PERV-A, -B, -C) was not observed, PERVs were expressed in all tissues, with the highest expression in the lung, followed by the ovary, blood, liver and placenta (Matousková et al., 2013). And a higher expression of PERV mRNA was found in melanomas than that in normal skin (Dieckhoff et al., 2007). It is of great interest that the integrated PERV proviral copy number differs among several organs of the same pig (Fiebig et al., 2018). As PERVs are transmitted vertically, like normal cellular genes, the number should be exactly the same in all organs of the same animal. These indicate that the expression and replication of PERVs are highly-regulated both temporally and spatially during pig development.
LTRs play a vital role in the regulation of the PERVs within the host genome. In the LTR U3 region, there are directly repeated nucleotide sequences. A majority of these repeat boxes exist as one of the two types, which are distinguished by a 39bp (PERV-A and -B) or 37bp (PERV-C) of the repeated nucleotides. And the multiplications of these repeat boxes also correlate with relatively late viral infection event, higher viral production, and higher infectious viral titer (Denner et al., 2003). After PERVs integrate into the host genome, these repeat boxes may increase the transcription of viral genes and flanked cellular genes. In response to the deleterious effects of the replicating PERVs, the number of these repeat boxes is restricted by natural instability and the constraints imposed by virion packaging limits (Łopata et al., 2018).
Heritable changes in PERV expression without changing the DNA sequence can occur through altering DNA methylation or histone modification. In mice, the endogenous retroviruses (ERVs) cause an estimated 10% of germline mutations in current inbred laboratory strains. Transcription of ERVs is generally repressed by epigenetic marks such as DNA methylation and histone modification (Schorn, Gutbrod, LeBlanc, & Martienssen, 2017). Likewise, treatment of cells with 5‑azacytidine, which promotes the demethylation of genomic DNA, results in PERV induction. It indicates the transcription of PERVs is sensitive to DNA methylation, and most PERV proviruses in pig genomes are currently silenced by DNA methylation on PERV 5’ LTRs CpG island (Matousková et al., 2013; Wolf, Nielsen, Mikkelsen, & Pedersen, 2013). And the PK-15 cell line, which has a very low DNA methylation level, keeps a high PERV transcriptional profile. And viral production also can exemplify the relation between the DNA methylation and the PERV mRNA transcription (Matousková et al., 2013).
Transcriptional silencing can also be achieved via establishment of a heterochromatic structure, which maintains an inactive form of chromatin. The highly condensed conformation prevents the access of transcriptional machinery and eventually suppresses transcriptional expression. Heterochromatin formation is associated with several histone marks in the genome, such as trimethylation of H3K9 (H3K9me3) and H4K20 (H4K20me3) (Lawrence, Daujat, & Schneider, 2016). Primarily the H3K9me3 has been found to be involved in inactivating viral chromatin of ERVs(Groh & Schotta, 2017). Meanwhile, knock-out experiments of methyltransferase, which generates the H3K9me3, have shed light on their involvement in ERVs silencing (Maksakova, Mager, & Reiss, 2008).
Likewise, PERVs are marked by the H3K9me3 and H4K20me3, and these marks were not restricted in porcine embryonic germ cells (pEGCs), but also apparent in PK15 cells. Interestingly, the younger and more active PERV subfamilies are generally marked by higher histone methylation levels than the ancient inactivated PERV subtypes (Wolf et al., 2013).