Long-read transcriptomics of Ostreid herpesvirus 1 uncovers a conserved expression strategy for the capsid maturation module and pinpoints a mechanism for evasion of the ADAR-based antiviral defence

Ostreid herpesvirus 1 (OsHV-1), a member of the family Malacoherpesviridae (order Herpesvirales ), is a major pathogen of bivalves. However, the molecular details of the malacoherpesvirus infection cycle and its overall similarity to the replication of mammalian herpesviruses (family Orthoherpesviridae ) remain obscure. Here, to gain insights into the OsHV-1 biology, we performed long read sequencing of infected blood clams, Anadara broughtonii , which yielded over one million OsHV-1 long reads. This data enabled the annotation of the viral genome with 78 gene units and 274 transcripts, of which 67 were polycistronic mRNAs, 35 ncRNAs and 20 natural antisense transcripts (NATs). Transcriptomics and proteomics data indicate preferential transcription and independent translation of the capsid scaffold protein as an OsHV-1 capsid maturation protease isoform. The conservation of this transcriptional architecture across Herpesvirales likely indicates its functional importance and ancient origin. Moreover, we traced RNA editing events using short read sequencing and supported the presence of inosine nucleotides in native OsHV-1 RNA, consistent with the activity of ADAR1. Our data suggests that, whereas RNA hyper-editing is concentrated in specific regions of the OsHV-1 genome, single nucleotide editing is more dispersed along OsHV-1 transcripts. In conclusion, we revealed the existence of a conserved pan- Herpesvirales transcriptomic architecture of the capsid maturation module and uncovered a transcription-based viral counter defence mechanism presumably facilitating the evasion of the host ADAR antiviral system. Author Summary Ostreid herpesvirus 1 (OsHV-1, family Malacoherpesviridae ) is a major pathogen of bivalve species, causing devasting mortalities and substantial economic losses of aquaculture species. The divergence of OsHV-1 compared to more extensively studied mammalian herpesviruses (family Orthoherpesviridae ) hampered the understanding of its biology. We performed a deep characterization of the OsHV-1 transcriptome based on long-read RNA sequencing produced from experimentally infected blood clams ( Anadara broughtonii ). Owing to the superior power of long read sequencing to disentangle overlapping transcript isoforms, we could reveal the complexity of the OsHV-1 transcriptome, composed of 274 transcripts. Despite the extensive divergence of OsHV-1 from vertebrate herpesviruses, we reported the presence of a pan- Herpesvirales transcriptomic architecture of the capsid maturation module, likely underpinning a conserved functional role in capsid assembly. Furthermore, we revealed the peculiar OsHV-1 transcriptomic patterns, presumably facilitating the evasion of the ADAR anti-viral defence system. In particular, OsHV-1 generates “molecular decoys” by co-expressing sense-antisense transcripts that sequester most ADAR RNA hyper-editing. Both these aspects support the existence of a functional role of “transcriptional architecture” in OsHV-1, contributing to a better understanding of the molecular behaviour of this virus. ### Competing Interest Statement The authors have declared no competing interest.