Replication and transcription machinery for ranaviruses: components, correlation, and functional architecture

Abstract

Background Ranaviruses (family Iridoviridae) are promiscuous pathogens that can infect across species barriers in poikilotherms and can replicate in amphibian and fish cells and even in cultured mammalian cells. However, as nucleocytoplasmic large DNA viruses (NCLDVs), their replication and transcription mechanisms remain largely unknown. Here, we screened and uncovered the replication and transcription machinery of two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV), by a combination of methods, including the isolation of proteins on nascent DNA, recombinant virus-based affinity, and NanoLuc complementation assay. Results The ranavirus replication and transcription machinery was deeply dissected and identified as a complicated apparatus containing at least 30 viral and 6 host proteins. The viral proteins ADRV-47L/RGV-63R (DNA polymerase, vDPOL), ADRV-23L/RGV-91R (proliferating cell nuclear antigen, vPCNA), ADRV-85L/RGV-27R (single-stranded DNA binding protein, vSSB), ADRV-88L/RGV-24R (vhelicase/primase), etc., constitute the core replisome. Specifically, the core of the transcription complex, the viral RNA polymerase, contain the host RNAPII subunits Rpb3, Rpb6, and Rpb11, which was a first report in NCLDVs. Furthermore, correlations and interactions among these factors in the machinery were described. Significantly, the replisome core protein vDPOL (ADRV-47L) can interact with numerous viral and host proteins and could act as a linker and regulation center in viral DNA replication and transcription. Thus, these results depicted an architecture for ranavirus replication and transcription. Conclusions Up to 36 components from ranavirus and their host were found to form viral replisomes and transcription complexes using a series of precise methods, which further constructed an architecture for ranavirus replication and transcription in which vDPOL was a key central factor and various components correlated and cooperated. Therefore, it provides a cornerstone for further understanding the mechanisms of the replication and transcription of ranaviruses which can ensure the efficient production of progeny virus and adaptation to cross-species infection.

ADRV 12L: A Ranaviral Putative Rad2 Family Protein Involved in DNA Recombination and Repair

“…ADRV 12L and its homologs (such as 95R of frog virus 3, and 102R of Rana grylio virus) have been considered as core genes of iridoviruses and were predicted as Rad2 family proteins, but their functions had not been investigated. In the present study, we cloned and characterized ADRV 12L as a virus encoded protein involved in DNA homologous recombination and repair, and it had been identified as a protein associated with viral nascent DNA in our previous study on the ranavirus replisome and transcription complex [ 20 ].…”

“…The total protein was collected at 0, 24, and 48 h post infection (hpi) for Western blot analysis as described above. In addition to the anti-ADRV 12L serum, previously prepared anti-ADRV 85L and anti-ADRV MCP serum [ 20 , 27 ] were used as controls.…”

“…For EdU labeling, EdU (Invitrogen, Carlsbad, CA, USA) was added to a final concentration of 10 μM at the indicated times. After a continued incubation for 30 min, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min, and then processed with a Click-iT Plus EdU Cell Proliferation Kit for Imaging (Thermo Fisher, Waltham, USA) according to the manufacturer’s protocols as described previously [ 20 ]. For antibody staining, EdU labeled or unlabeled cells were incubated with anti-ADRV 12L or anti-ADRV 85L serum at a dilution of 1:100 as the primary antibody.…”

“…As large DNA viruses, ranaviruses have been predicted to encode several enzymes involved in DNA replication and transcription, such as the DNA polymerase, RNA polymerase subunits, and some accessory proteins. In previous work, we screened the proteins involved in virus replication and transcription in ADRV infected cells, in which ADRV 12L was found [ 20 ]. In the present study, we cloned and analyzed the function of ADRV 12L by subcellular localization, knockout mutant virus, and homologous recombination (HR) assays.…”

Andrias davidianus Ranavirus (ADRV) Genome Replicate Efficiently by Engaging Cellular Mismatch Repair Protein MSH2

“…Because of the intracellular life cycle, virus replication involves not only viral proteins but also host proteins. In a recent study on the replication and transcription machinery of ranaviruses, we identified several viral and host proteins as potential proteins associated with viral nascent DNA by proteomic analysis of ADRV infected cells [ 14 ]. Among them, the host MutS homolog 2 (MSH2) was found but its function in virus infection was unknown.…”