Members of the genus Ranavirus (family iridoviridae) are nucleo-cytoplasmic large DNA viruses (NCLDVs) that are isolated from aquatic animals including reptiles, amphibians, and bony fish. They have been considered as promiscuous pathogens that pose a great threat to economically and ecologically important poikilotherms. Many ranaviruses can infect more than one host species and cross species barriers, yet mechanisms on renavirus genome replication and transcription are remained largely unknown.   

The research group led by Prof. ZHANG Qiya from the Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences dissected the components of the replication and transcription machinery for ranaviruses, and identified the correlations and functional architecture of these components. The study was published in Cell & Bioscience.   

In this study, the researchers first examined 5-ethynyl-2'-deoxyuridine (EdU) labeling and its conditions in two ranaviruses - Andrias davidianus ranavirus (ADRV) or Rana grylio virus (RGV), which infected the Chinese giant salamander thymus cells (GSTC), and confirmed that iPOND (isolation of proteins on nascent DNA) could be applied efficiently in lower vertebrate cells.    

Using the iPOND coupled with Mass spectrometry, they screened a total of 46 ADRV proteins, 38 RGV proteins, and several host proteins, which are viral nascent DNA associated proteins, in each virus infected GSTC cells.    

By in vitro protein purification and electrophoretic mobility shift assay, the researchers then identified the ranavirus encoded single-stranded DNA binding (SSB) proteins as shown in a pair of homologue proteins from the two ranaviruses, ADRV-85L and RGV-27R, which exhibited high abundances in above iPOND-MS assays.    

Immunofluorescence and co-immunoprecipitation (co-IP) were used to further analyze the colocalization and interaction of the viral replisome core components - viral encoded helicase/primase, SSB, DNA polymerase, and processivity factor. The results indicated the complex interactions among the four proteins.   

A new established NanoLuciferase (NanoLuc) complementation assay in aquatic animal cells has shown that the viral DNA polymerase interacted with a number of viral proteins, as proved by co-IP. The interacted proteins included not only the proteins involved in DNA replication but also these involved in transcription, which hinted a central function of the DNA polymerase.   

In addition, the researchers found that host topoisomerases IIα and IIβ are core viral replisome components by using specific inhibitor, immunofluorescence, and co-IP. The two proteins interacted with viral proteins, and located in the cytoplasmic viral factories from nucleus during virus infection.   

Through recombinant virus based affinity purification and Mass spectrometry, they also identified the proteins associated with viral transcription machinery, which contained eight viral proteins and three host proteins. The three host proteins are the host RNA polymerase II subunits Rpb3, Rpb6, and Rpb11, suggesting that host RNA polymerase subunits were hijacked by virus. The interactions between the three host proteins and viral proteins were proved by followed co-IP assays.   

This study was the first to shed light on the complicated replication and transcription machinery of ranaviruses by using two ranaviruses. It also offers insights into understanding the efficient replication and transcription of ranaviruses, and into the interactions between ranaviruses and their hosts.