Evolution of ERV LTR sequences. (A) The LTRs of an ERV are identical at the time of integration. Diagram D1, hypothetical provirus with spontaneous LTR mutations at points A, B, C, and D. Diagram D2, a newly transcribed viral RNA genome will contain only the mutations at points B and C. Diagram D3, after reverse transcription and integration, the new provirus has identical LTRs with mutations B and C found in both LTRs. (B) Hypothetical phylogeny based on ERV LTR sequences. The tree contains four distinct types of substitutions. 5′ and 3′ LTRs from the same provirus are expected to cluster separately, because of substitutions that predate all speciation events (“1”). The node joining the two clusters represents the time of integration, when the two LTRs were identical. Substitutions that occur between speciation events (synapomorphies) fall exclusively within one or the other cluster and define the topology of the cluster according to the evolutionary history of the different species (“2”). Assuming that both LTRs have evolved independently and at the same rate, the two clusters will have the same branching pattern, revealing the phylogeny of the input species. Substitutions unique to one species (autoapomorphies) are phylogenetically uninformative (“3”). Branches separating distinct ERV loci, e.g., between the ingroup provirus HERV 1 and the outgroup provirus HERV 2, represent errors accumulated during viral replication (“4”). Thus trees containing sequences from more than one HERV can be rooted at the node joining the two proviruses, because the two loci share a common viral ancestor. Rooting the tree with another ERV is therefore independent of any assumptions about host species phylogeny. (C) PCR amplification of ERV LTRs from genomic DNA. Arrows indicate 5′ → 3′ orientation of PCR primers; thick lines, cellular flanking sequences; thin lines, ERV sequences; boxes, LTR sequences. LTRs and adjacent cellular sequences are amplified by using one flanking sequence-specific primer and one provirus-specific primer (depicted as primers 1 and 2 for the 5′ LTR and primers 3 and 4 for the 3′ LTR). If neither LTR can be detected the presence of the uninterrupted cellular sequence or solo LTR can be determined by using primers 1 and 4. Proviral integration is essentially random, so flanking sequences amplified with each LTR confirm that homologous loci are being compared. Integration also results in a duplication of target sequences (4–6 bp) flanking each provirus, which can be used to confirm that the amplified 5′ LTR and 3′ LTR are from the same provirus.