Functional genomics studies of large DNA viruses have been facilitated by the construction of infectious bacterial artificial chromosomes , which supply a useful molecular manipulation platform for serial knock out and knock in of viral genes and examination of the subsequent effects on viral infection [11, 12, 36, 41], such as been done for human hepersviruses and baculoviruses. With increase in the number of sequenced ascoviridae genomes, identification of Acoviridae orthology and protein phosphorylation is expected to facilitate an understanding Acoviridae phylogenomics and functional genomics which has been hindered by a lack of constructed bacterial artificial chromosomes for the molecular manipulation of Ascoviridae species.
The new annotation of 90 proteins missing in previous annotated genomes facilitates understanding of the protein conservation among Ascoviridae genomes, and the comprehensive identification of 366 homologous groups supplies a checklist for future annotation of Ascoviridae genomes. In a comprehensive view, most of the core genes commonly found in Ascoviridae species or Ascovirus species are single-copy and orthologous genes as well. Nevertheless, the orthology of non-single copy core genes needs further differentiation based on phylogenetic inference of gene/protein trees. This prompted the first construction of a phylogenetic tree of Ascoviridae species using concatenated orthologous genes in this study, rather than using individual genes [14, 40, 50].
Conserved genes seem to be important for viral infection process, such as viral DNA replication, RNA transcription and viral regulation by host (signaling and protein-protein interaction network) and these facilitate virus reproduction. Among the 45 core genes in HvAV-3h, 27 proteins were involved in DNA/RNA replication/transcription/metabolism, packaging and assembly, signaling, sugar and lipid metabolism, and cell lysis (Table 1), while the involvement of viral proteins in viral entry into a host cell is unknown. Apart from the membrane proteins of Cathepsin B (ORF109), Lysophospholipid acetyltransferase (ORF113), Patatin-like phospholipase (ORF133), and Fatty acid elongase (ORF146), which can facilitate formation and release of viral vesicles , the five HvAV-3h proteins including ORF48 (486aa), Myristylated membrane protein-like protein (ORF62) (292aa), ORF64 (427aa), ORF118 (168aa), ORF134 (219aa) conserved in Ascoviridae genome, and three proteins including ORF55 (225aa), ORF69 (78aa) and ORF128 (268aa), were conserved among Ascovirus genome, and were screened out as conserved membrane proteins with unknown functions. Taking protein conservation and association with virions into consideration , the involvement of these membrane proteins in viral entry needs to be further elucidated.
Protein phosphorylation targeted by various kinases are usually involved in regulation of protein function, cellular location and interaction with other proteins . Of the 175 phosphorylation sites identified here, 89 sites and their surrounding motifs were matched with consensus motifs targeted by eukaryotic kinases, especially by the CK1 CAMK2, and PKA kinases, and the eukaryotic kinase CK1 targeted six conserved phosphorylation sites, indicating the involvement of eukaryotic kinases in the phosphorylation of viral proteins. Furthermore, the presence of more phosphorylation sites with no matched motif targeted by eukaryotic kinases will enrich the set of known phosphorylation motifs of kinases . For the phosphoproteins conserved among all Ascoviridae species, HvAV-3h ORF50 of 189 amino acids in length was highly phosphorylated with 10 phosphorylation sites, and contained five conserved phosphorylation sites including one tyrosine site (Y126), supplying a potential research target for investigation of a protein of unknown function. In addition, two serine/threonine protein kinases were found among all Ascoviridae species and need further work to unveil their involvement in the phosphorylation of host and viral proteins .
In sum, this study updated our understating of Ascoviridae protein conservation and species phylogeny based on a comprehensive identification of protein orthologs from homologue groups and phylogenetic analysis of non-single copy proteins, and provides multiple phosphorylation sites, in particular the conserved sites for virion-associated phosphoproteins. This should facilitate future annotation of increasing numbers of Ascoviridae genomes and lays a foundation and identifies potential research targets for further understanding of virus infection mechanism especially viral entry into host cells, despite the present circumstances of limited function genomic analysis of Ascoviridae species due to lack of molecular manipulation platforms.