Nucleo-cytoplasmic large DNA viruses (NCLDVs) of the genus Ranavirus (family iridoviridae) have been isolated from aquatic species such as reptiles, amphibians, and bony fish. Poikilotherms are at risk from these promiscuous infections.
Despite the fact that many ranaviruses may infect several host species and traverse species boundaries, the processes governing renavirus genome replication and transcription are mostly unexplored.
Prof. Qiya Zhang of the Chinese Academy of Sciences, Institute of Hydrobiology (IHB) examined the components of the replication and transcription machinery for ranaviruses, identifying the connections and functional architecture of these components.
This study was published on January 6th, 2022, in the journal Cell & Bioscience.
The researchers looked at the circumstances of 5-ethynyl-2’-deoxyuridine (EdU) labeling in two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV) that infected the Chinese giant salamander thymus cells (GSTC).
Scientists screened 46 ADRV proteins, 38 RGV proteins, and many host proteins which are viral nascent DNA associated proteins, in each virus-infected GSTC cell using isolation of proteins on nascent DNA (iPOND) combined with mass spectrometry.
The ranavirus encoded single-stranded DNA binding (SSB) proteins were identified by researchers as shown in a homolog proteins pair from the two ranaviruses: ADRV-85L and RGV-27R, which equipped greater abundances in the above iPOND-MS assays.
The co-localization and interaction of the viral replisome core components—viral-encoded helicase/primase, SSB, DNA polymerase, and processivity factor were studied further using immunofluorescence and co-immunoprecipitation (co-IP). The study indicates the four proteins’ intricate connections among each other.
The viral DNA polymerase interacted with several viral proteins, as demonstrated by co-IP, in a newly created NanoLuciferase (NanoLuc) complementation test in aquatic animal cells. The proteins that interacted with each other included not only the proteins involved in DNA replication but also those involved in transcription, suggesting that the DNA polymerase has a vital function.
Using a particular inhibitor, immunofluorescence, and co-IP, the researchers discovered that host topoisomerases IIα and IIβ are major viral replisome components. During virus infection, the two proteins interacted with viral proteins and were found in the cytoplasmic viral factories from the nucleus.
Scientists also discovered the viral transcription machinery proteins, which included eight viral proteins and three host proteins. The three host proteins are the Rpb3, Rpb6, and Rpb11 subunits of host RNA polymerase II, implying the virus captured the host RNA polymerase components. Co-IP tests were used to confirm the interactions between the three host proteins and the viral proteins.
Using two ranaviruses, this work was the first to shed new light on the intricate replication and transcription machinery of ranaviruses.
Ke, F., et al. (2022) Replication and transcription machinery for ranaviruses: components, correlation, and functional architecture. Cell & Bioscience. doi.org/10.1186/s13578-021-00742-x