Human cytomegalovirus harnesses host L1 retrotransposon for efficient replication

Given the mobility of L1 and the involvement of L1 ORF2p in HCMV replication, we investigated whether endogenous L1 retrotransposition facilitates the genetic adaptation of HCMV during lytic infection. If certain L1 insertions were to promote the fitness of carrier HCMV, these insertions would be expected to be inherited by progeny viruses and amplified via clonal expansion in the virus population. To test this hypothesis, we first applied human active transposon-sequencing (HAT-seq), a PCR-based L1 capture method, to profile L1 insertions in the HCMV genomes. We were unable to identify high-confidence clonal L1 insertions integrated into the HCMV genomes. We additionally performed ultra-deep Illumina whole-genome sequencing (WGS) to comprehensively identify all forms of L1-mediated retrotransposition, including Alu and SVA, in the HCMV genome. We found no evidence of putative clonal transposon insertions with an allele frequency greater than 0.1% in the viral population.Since HCMV exhibits high genetic diversity during natural infection, we determined whether L1-mediated DNA damage leads to accumulated mutations in the HCMV genome, thereby conferring enhanced viral replication fitness. To this end, we investigated the underlying patterns of L1-mediated mutagenesis within the viral genomes of WT, shLacZ, and shL1 HCMVs by revisiting deep Illumina short-read WGS of HCMV. There was no overall significant enrichment of mutations across WT and shLacZ samples compared with shL1 samples.