Insight Into The Virophages And Their Potential Hosts(CVV System) In Dishui Lake

Viophage is a small double-stranded DNA virus.The mature virus particles of virophage are not encapsidated but consisting of an icosahedral nucleocapsid with the diameter of 35-74 nm and a genome of 16-19 kb length encoding 16-34 genes.Virophage cannot independently reproduce within the eukaryotic host,and it must rely on the virus factory formed by giant virus.What's more,virophage can cause morphological deformity and virulence declining of giant virus,and even can package its mature virus particle into the capsid of giant virus which like infecting its host virus.Analog the bacteriophage,then these kinds of virus are collectively referred to as virophage.In the infection system composed of eukaryotic cells,host virus and virophage,we call the CVV system,the virophage and its host virus play a crucial role to understand the nature of virus by their special interacts in infection strategy,reproducing mode,genetic content,evolutionary relationship,immune mechanism and other aspects.Unfortunately,although there are evidences that virophage may have a global distribution,only 18 complete genomes of virophage and three culturable CVV systems have been obtained so far.Are there any other cells that can form a new CVV system? In recent years,the study of the new virophages has given the potential of the new CVV system which is composed of green algae and giant phycodnaviruses.Based on the previous studies,this paper further explores a new CVV system in algae cell.We mainly explored the genome contents and genetic interaction of novel virophages and their potential virus host,i.e.phycodnaviruses by insight into the metagenomic database of Dishui Lake.Meanwhile,we tried to reveal the relationship between the new virophages and their eukaryotic host at the protein level.In addition,we attempted to construct a new CVV system which is composed of green algae isolated from DSL,phycodnaviruses and virophages.The main results of this paper are presented as following.(1)The whole genome sequence of four novel virophages were obtained.Through a lot of exploration in the sequence assembly,we finally get four new virophage whole genome sequences from the DSL metagenomic dataset.They are named Dishui Lake Virophage 3/4/5/6(DSLV3/4/5/6),and the sequence length of them are 31,512 bp,30,873 bp,26,593 bp,28,714 bp,respectively.All these virophage genome harbor a circular genome except DSLV3.In addition,palindromic repeats were found at the asymmetric position of two ends in the DSLV3 genome,the same as the previously find in the giant virus genome,which may indicate that DSLV3 possess a unique genomic structure.Phylogenetic analysis revealed that these novel virophages could form a monophyletic group with Yellowstone Lake Virophage(YSLVs)and Antarctic Organic Lake Virophage(OLV),but independent of the known virophages in the three CVV systems,e.g.Sputnik,Mavirus and Zamilon.Comparative genomic studies revealed that there are some homologous genes in DSLVs from phycodnaviruses,algal RNA viruses and eukaryotic algae.The discovery of these homologous genes indicates the horizontal gene transfer between these novel virophages and their host systems.(2)The whole genome sequence of four novel phycodnaviruses were obtained.Since the phycodnavirus genes were found in virophages genome,we start to focus on the phycodnaviruses in DSL metagenomic database.After a lot of explores on sequence assembly,we finally obtained four novel phycodnavirus genomes.They are named as Dishui Lake Phycodnavirus 2/3/4/5(DSLPV 2/3/4/5).The sequence length of them were 190,426 bp,194,169 bp,392,531 bp,193,666 bp,respectively.All of them harbor a linear genome except DSLPV4.Phylogenetic analysis revealed that these phycodnaviruses originating from freshwater environments were closely related to the marine algal prasinovirus,which broadened our understanding of the diversity of phycodnavirus.Above all,some virophage and algal homologous genes were detected in DSLPVs genome as well which consistent with the findings in the genome of virophage.These evidences further indicating the possibility of a new CVV system in the Dishui Lake ecosystem with DSLVs,DSLPVs and eukaryotic algae.(3)Monoclonal antibodies of the major capsid protein(MCP)from DSLV3 were obtained.Through the construction of p GEX-4T1-MCP expression system,we successfully get virophage capsid protein in the prokaryotic expression system for the first time,but subsequent experiments showed that this protein belongs to the inclusion body rather than the supernatant protein we needed.Then we tried to optimize the expression system to get the supernatant protein,but it failed.To avoid affecting the subsequent test progress,we finally give up the expression work of the protein and transferred this work to the Shanghai biotechnology company.Fortunately,we eventually got the antibody,which provide support for the follow-up experiment.(4)A kind of microalge Mychonastes afer was obtained.Based on our previous work,we have made a general understanding of the distribution of algae in the Dishui Lake.Combined with the above genomic analysis,we have assumed that there is a novel CVV system composed of green algae(especially Prasinophytes),phycodnavirus(DSLPVs)and virophage(DSLVs)in Dishui Lake ecosystem.To verify this hypothesis,we try to isolate some kinds of green algae in the Dishui Lake.Through the plate striking and spreading,we luckily get one species of green algae(algae isolation and purification work is still in progress).Molecular identification and morphological analysis confirmed that it is Mychonastes afer which is close relatives of Prasinophytes.We will use the pure culture of the isolated algae as the model cell to enrich the phycodnavirus and virophage and establish the CVV infection system in the future.Fortunately,we obtained a complete set of protocols of algae isolation,purification in the whole experimental process,which provide a guidance for the follow-up experiments.