The Number of a U3 Repeat Box Acting as an Enhancer in Long Terminal Repeats of Polytropic Replication-Competent Porcine Endogenous Retroviruses Dynamically Fluctuates during Serial Virus Passages in Human Cells

Abstract

The organization and transcriptional regulation of porcine endogenous retrovirus (PERV) long terminal repeats (LTRs) are unknown. We have studied the activity of LTRs from replication-competent molecular clones by performing luciferase reporter assays. The LTRs differ in the presence and number of 39-bp repeats located in U3 that confer strong promoter activity in human, simian, canine, feline, and porcine cell lines, whereas for LTRs devoid of the repeats, the promoter strength was significantly reduced. As the activity of a heterologous simian virus 40 promoter and a homologous repeat-deficient LTR was elevated by four 39-bp repeats independently of its orientation and location, the repeat box complies with the definition of an enhancer. During serial virus passaging of molecular PERV clones on human 293 cells, proviral LTRs demonstrated adaptation of transcriptional activity by dynamic changes of the number of 39-bp repeats in the course of up to 12 passaging cycles.

Evolutionary Spread and Recombination of Porcine Endogenous Retroviruses in the Suiformes

“…PERV-A and PERV-B proviruses demonstrate LTRs that harbor distinct 39-bp repeats in U3 which enable high transcription levels and adaptation to new host cells by multimerization of these repeats, with the transcriptional activity being generally stronger if more repeats are present. On the other hand, PERV-A and PERV-C were found to display repeatless LTRs (37,47). Although sequences homologous to the 39-bp repeat are present in these LTRs, they are not organized in a repetitive manner and do not show multimerization as a response to replication cycles in their hosts.…”

“…There are only minor genetic differences between the classes, being most prominent in the receptor-binding domain of the Env protein. In addition, there are two different types of long terminal repeats (LTRs) that significantly affect the replication properties of single viruses (37,47) through binding of transcription factor NF-Y (36). PERV-A and PERV-B proviruses demonstrate LTRs that harbor distinct 39-bp repeats in U3 which enable high transcription levels and adaptation to new host cells by multimerization of these repeats, with the transcriptional activity being generally stronger if more repeats are present.…”

Methods and Tools for Detection and Evaluation of the Risks of Porcine Endogenous Retrovirus in Porcine to Human Xenotransplantation

“…While our own study did not show significant differences in expression in different cell lines tested for LTRs derived from PERV-A, -B, or -C, we did see a slightly lower level of expression with a chimeric LTR called PERV-NIH and all LTRs tested showed lower expression levels in ST-IOWA pig cells compared to other cell lines examined (Wilson, Laeeq et al 2003). In other studies, it has been shown that the level of PERV expression in permissive cells is associated with the copy number of a 39 base pair (bp) repeat in the PERV LTR U3 region (Scheef, Fischer et al 2001). A more thorough study of the LTR structures from kidney of NIH minipigs has shown that there are four different elements and that one was associated with the highest level of transcriptional activity.…”

Promoter activity analysis and methylation characterization of LTR elements of PERVs in NIH miniature pig

“…Tandem repeat sequences, in particular, are known to affect the promoter or enhancer activities of LTR elements (Scheef et al, 2001). A 39-bp repeat sequence exists in the U3 region: it comprises a tandem repeat that contains 2 of the original 18-bp repeat and original 21-bp repeat sequences.…”

“…Generally, the R and U5 regions are conserved sequences. PERV-A and B proviruses contain distinct 39-bp repeats in the U3 regions, which enable PERVs to maintain a high level of transcription (Scheef et al, 2001). Thus, mutations such as deletions, insertions, and nucleotide substitutions could transmute the transcriptional activity of a PERV element.…”