Genomic Characterisation of Chromosomally Integrated Human Herpesvirus

Distinctions in the biological features of human herpesvirus-6A (HHV-6A) and -6B (HHV-6B) have recently led to their reclassification from variants to distinct viral species. Unique for human herpesviruses, these viruses also exist in an endogenous form in ~1% of the human population, resulting from germ-line chromosomal integration. Chromosomally integrated human herpesvirus-6A (CI-HHV-6A) and -6B (CI-HHV-6B) genomes are inherited in a Mendelian manner, leading to the potential for viral gene expression and/or reactivation in every cell of the body. As yet, the effects of these integrated viral genomes on health and their relationship to circulating viral strains remain unclear. To address this, next generation sequencing (NGS) methods were established, to define a readily utilisable pipeline for genomic sequencing of these herpesviruses directly from cell or tissue samples. This was first tested to derive the complete genome sequence of a third HHV-6A strain, AJ. This showed close conservation with a recent North American isolate despite distinct geographic origins, which may reflect highly evolved viral status or recent emergence. Subsequently, using this defined NGS methodology, supplemented by Sanger sequencing, integrated viral genomes were characterised. Integration site analyses revealed a high prevalence of integration on chromosome 17, all with a shared junctional architecture, indicative of a single ancestral germ-line integration event at this chromosome. Genomic analyses revealed CI-HHV-6A retains the full HHV-6A gene complement, with no interrupted open reading frames, and important cis acting signals likely required for any viral replication/reactivation mechanism. Deep sequencing with minority variant analysis suggested a potential reactivation mechanism, resulting from superinfection with circulating viral strains. CI-HHV-6A was also found to diverge at a set of genes which have been used as markers of speciation amongst roseoloviruses, while retaining many of the features of HHV-6A. One of the key viral immunomodulatory genes, the chemokine receptor U51, was investigated further. This showed specific coding changes and a panel of viral-human chimeric and viral mutant receptor expression vectors were generated to investigate this. Initial structural modelling and functional characterisations were made showing distinctions. Overall, the results identify unique characteristics of the integrated genomes compared to known circulating viral strains.